Compositions and methods for inducing humoral and cellular immunities against tumors and cancer

ABSTRACT

Provided are methods for sensitizing gastrin-associated tumors and/or cancers in subjects to inducers of humoral and cellular immune responses. In some embodiments, the methods relate to administering compositions that have anti-gastrin antibodies, gastrin peptides, and/or nucleic acids that inhibit expression of gastrin gene products to subjects. Also provided are methods for preventing, reducing, and/or eliminating the formation of fibroses associated with tumors and/or cancers, and methods for treating gastrin-associated tumors and/or cancers that include administering to subjects in need thereof a first agent that provides and/or induces an anti-gastrin humoral or cellular immune response in the subject and a second agent that includes one or more stimulators of cellular immune responses against the tumors and/or cancers.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/520,267, filed Jun. 15, 2017, the disclosure ofwhich is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The presently disclosed subject matter relates to compositions andmethods for inducing both humoral and cellular immunities against tumorsand cancers. In some embodiments, the presently disclosed subject matterrelates to administering to a subject in need thereof a therapeuticinducer of a humoral or cellular immune response against a gastrinpeptide and/or in combination with an inducer of a cellular immuneresponse against the tumor or the cancer.

BACKGROUND

Pancreatic cancer, generally referred to as pancreatic ductaladenocarcinoma (PDAC) is a complex disease involving the successiveaccumulation of genetic mutations in several cell growth regulatorypathways. What begins as relatively benign lesions in a pancreaticintraepithelial neoplasia (PanIN; Hruban et al., 2008) progresses into adiversity of abnormal gene expression patterns, genomic instability, andultimately invasive cancer that is resistant to treatment.

Histologically, PDAC is generally well-differentiated and is primarilydefined by acinar-ductal metaplasia, the presence of immunosuppressiveinflammatory cells, lack of cytotoxic T-cells, and the presence of adense fibrotic stroma. These manifestations can vary greatly in extentand can occur without overt clinical symptoms, which makes earlydiagnosis of PDAC a rarity. The PDAC tumor stroma consists ofmesenchymal cells such as fibroblasts and pancreatic stellate cells(PSCs), extracellular matrix proteins, peri-tumoral nerve fibers,endothelial cells, and immune cells. The specific mechanisms influencingthe stromal cells to produce the abundant desmoplastic effects involvegrowth factor activation, collagen and extracellular matrix synthesisand secretion (Zhang et al., 2007), as well as the expression ofnumerous regulators of vascular and cytokine-mediated processes (Hidalgoet al., 2012).

Invasive PDAC constitutes the vast majority (>85%) of carcinomas ofductal lineage. PDAC is characterized by uncontrolled infiltration andearly metastases. The presumed precursors of ductal adenocarcinoma arethe PanIN microscopic lesions that undergo intraductal proliferativechanges and ultimately a series of neoplastic transformations fromPanIN-1A to PanIN-3 and full-blown malignant carcinoma.

Important characteristics of PDAC are aberrant expression of thegastrin/cholecystokinin receptor (CCK-B) on the surface of tumor cells(Smith et al., 1994) as well as the expression of high levels of gastrinby the tumor (Prasad et al., 2005). Both gastrin (Smith, 1995) andcholecystokinin (Smith et al., 1990; Smith et al., 1991) proteinsstimulate pancreatic tumor growth through an autocrine mechanism (Smithet al., 1996a; Smith et al., 1998b), and inhibition of either gastrinexpression (Matters et al., 2009), or blockage of CCK-B receptorfunction (Fino et al., 2012; Smith & Solomon, 2014) inhibits cancergrowth.

In spite of impressive success in the treatment of many cancers over theyears, tragically there has been little to no success in the marketapproval of breakthrough therapeutics for PDAC (see Hidalgo, 2010; Ryanet al., 2014), which carries the poorest prognosis of allgastrointestinal malignancies (Siegel et al., 2016). The currentfive-year survival rate for PDAC is approximately 6%, the lowest of anycancer (Siegel et al., 2016).

The poor outcome of PDAC has not changed for the past 30 years. Amultidisciplinary diagnosis followed by surgery and chemo- and radiationtherapy is the first-line treatment approach. However, therapies basedon the small molecule chemotherapeutics gemcitabine and 5-fluorouracildo not produce satisfying outcomes and mean survival with these regimensremain less than 1 year (Hoff et al., 2011, Conroy et al., 2011).

Contributing factors to the poor survival rates include the inability todiagnose this disease in the early stages, the heterogeneity of cellularand anatomical tumor cells, the high rate of metastasis, and thepresence of a dense fibrotic microenvironment that inhibits drugpenetration and exposure (Neesse et al., 2013). Inaccessibility of thetumor results in a relative resistance of PDAC to standard chemotherapyand immunotherapy agents (Templeton & Brentnall, 2013) and contributesto the poor prognosis for this fatal disease.

The host immune response is another key factor contributing to therecalcitrant and aggressive nature of PDAC. Immune cells, which are soprominent in the microenvironment of PDAC, do not support anti-tumorimmunity (Zheng et al., 2013). Rather, these cells (includingmacrophages, T-regulatory (T_(reg)) cells, and neutrophils), actuallypromote tumor growth and invasion. In fact, one of the hallmarks of PDACis its ability to evade immune destruction (Hanahan & Weinberg, 2011).

Cancers, including PDAC, employ many tools to escape and/or defeatattack from the patient's immune system (Pardoll, 2012; Weiner & Lotze,2012). Components of the tumor metabolic milieu have been shown toregulate these responses (Feig et al., 2012; Quante et al., 2013). Amajor breakthrough in cancer therapeutics came with the discovery ofimmune checkpoint pathways that are often regulated by tumor cells as amechanism of immune resistance (Leach et al., 1996). Antibodies thattarget proteins in the checkpoint pathways, such as cytotoxic Tlymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein1 (PD-1), and programmed cell death ligand 1 (PD-L1), have beendeveloped and have been shown to be clinically effective in reversingimmunoresistance in some cancers, such as melanoma, non-small cell lungcarcinoma (NSCLC), and renal cancer (Pardoll, 2012). However, PDAC ischaracterized by a tumor microenvironment that has a predominance ofimmune-suppressing T regulatory (T_(reg)) cells, lacks CD8⁺tumor-infiltrating effector T cells (Feig et al., 2012; Vonderheide &Bayne, 2013; Zheng et al., 2013), and is poorly vascularized. Thefibrotic nature of the dense stromal environment as well as the lack ofaccessibility through the bloodstream explains in part the observationthat PDAC responds only modestly, at best, to anti-PD-1 and anti-PD-L1antibodies (Brahmer et al., 2012, Zhang, 2018).

The expression level of the checkpoint ligand PD-L1 on the surface ofPDAC cells is believed to be another determinant of response to immunecheckpoint inhibitor immunotherapy (Zheng, 2017). Some studies havesuggested that a low level of PD-L1 expression correlates with the lackof response to immune checkpoint inhibitors (Soares et al., 2015), andthat stimulation of PD-1 or PD-L1 expression can help to facilitate theeffectiveness of anti-checkpoint protein antibodies (Lutz et al., 2014).In other studies of PDAC, PD-L1 was found to be highly expressed in amajority of tumor cells as well as in many tumor samples (Lu et al.,2017). Thus, the effectiveness of immune checkpoint inhibitor therapycould potentially be enhanced by considering the status of PD-L1 in thetumor and in seeking methods for regulating PD-L1 expression toaccompany PDAC-targeted therapy.

Currently, clinical trials for treatment of PDAC include combiningantibody immune checkpoint inhibitors with chemotherapy, radiation,chemokine inactivation (olaptesed), cyclin dependent kinase inhibition(abemaciclib), TGF-β Receptor I kinase inhibitors (galunisertib), focaladhesion kinase inhibitors (defactinib), CSF1R inhibitors(Pexidartinib), vitamin D, and Poly ADP ribose polymerase inhibitors(niraparib). These studies are aimed at combining agents that mightimprove the physical penetration of and/or the immune cell presence inthe PDAC tumor microenvironment, as well as to improve the effectivenessof immune checkpoint inhibitor treatment. In a recent report (Smith etal., 2018), inhibition of CCK-B receptor function reduced PDAC fibrosisand improved the effectiveness of antibody therapy using an anti-PD-1antibody (Ab) and an anti-CTLA-4 Ab.

Given the complexity of the PDAC tumor, a deeper understanding is neededof how novel strategies can be used to modify the immune phenotype ofthe PDAC microenvironment across the heterogeneity of patients and tomake the tumor more responsive to both chemo- and immune-basedtherapies.

Gastric cancer is another devastating cancer, and gastric adenocarcinomain particular has one of the poorest prognoses of all cancers, with a5-year survival of up to 30% (Ferlay et al., 2013). Early detection ofthis malignancy is elusive and requires intentional screening practices,which are not commonly utilized. Most diagnoses are already in advancedstage with median survival of 9-10 months (Wagner et al., 2010; Ajani etal., 2017). The current standard of care for gastric cancer includessurgery when appropriate, followed by radiation and/or chemotherapy withDNA synthesis inhibitors like 5-fluorouracil and/or DNA damaging agentssuch as cis-platinum.

Targeted therapies have also begun to emerge for the treatment of somegastric cancers. Tumors that express the human epidermal growth factorreceptor 2 (EGFR2) can be treated with trastuzumab (sold under thetradename HERCEPTIN® by Genentech, Inc., South San Francisco, Calif.,United States of America) in combination with chemotherapy. Some gastriccancers are also responsive to anti-angiogenesis drugs such asramucirumab (sold under the tradename CYRAMZA® by Eli Lilly and Company,Indianapolis, Ind., United States of America). Additional targetedtherapies are urgently needed to improve the dismal prognosis for thisprevalent malignancy.

Gastric adenocarcinomas typically overexpress gastrin as well as thereceptor for gastrin, called the CCK-B receptor (Smith et al., 1998a;McWilliams et al., 1998), and gastrin-mediated proliferative effectsupon binding to CCK-B lead to an uncontrolled autocrine cycle of growthand expression in these tumors. Blocking the function of gastrin as ameans of therapy for this cancer has been a focus of research for manyyears (reviewed in Rai et al., 2012). Among the candidates for targetedtherapy, the gastrin vaccine Polyclonal Antibody Stimulator (PAS) hasshown significant promise in improving survival in gastric cancer inPhase 2 clinical trials and in pancreatic cancer in Phase 2 and Phase 3clinical trials. PAS vaccination has been shown to elicit a humoralimmune response as demonstrated by the production of neutralizingantibodies to gastrin. By eliminating gastrin, the vaccine slows tumorgrowth and has potential to provide long-term tumor killing activity.

Cancer vaccines that raise an immune response against specific tumorantigens are an attractive treatment strategy when the immune-mediatedimmobilization or inactivation of the target antigen does not havedeleterious effects elsewhere in the body. Peptide vaccines have thepotential advantage of narrowing the specificity of the immune response,but they can sometimes have the disadvantage of eliciting a weakimmunogenicity. Careful selection of peptide composition as well asincorporation of adjuvant molecules and delivery systems can benecessary to insure a robust response as well as to initiate inductionof the desired immunity pathway. Peptides as short as 9-11 amino acidscan generate a specific CD8+ T cell-mediated response, though a changeof even one amino acid in the epitope can prevent the response (Gershoniet al., 2007).

The choice of epitopes to be included on the peptide requires theconsideration of type of immune response desired, including MHC class IIepitopes to induce CD4+ helper T cells and MHC class I CD8 epitopes toinduce helper T cells and CD8+ cytotoxic T lymphocytes (Li et al.,2014).

The combination of a gastrin peptide vaccine, such as PAS, combined withan immune checkpoint inhibitor represents a novel approach to improvingoutcome in cancers that are subject to growth stimulation by the gastrinpeptide hormone.

SUMMARY

This Summary lists several embodiments of the presently disclosedsubject matter, and in many cases, lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

In some embodiments, the presently disclosed subject matter providespharmaceutical compositions comprising a first agent that induces and/orprovides an active and/or a passive humoral immune response against agastrin peptide and/or a CCK-B receptor and an immune checkpointinhibitor. In some embodiments, the first agent is selected from thegroup consisting of a gastrin peptide, an anti-gastrin antibody, and ananti-CCK-R antibody. In some embodiments, the first agent comprises agastrin peptide, optionally a gastrin peptide comprising, consistingessentially of, or consisting of an amino acid sequence selected fromthe group consisting of EGPWLEEEEE (SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO:2), EGPWLEEEEEAY (SEQ ID NO: 3), and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4).In some embodiments, the glutamic acid residue at amino acid 1 of any ofSEQ ID NOs: 1-4 is a pyroglutamate residue.

In some embodiments, the gastrin peptide is conjugated to an immunogeniccarrier, optionally via a linker. In some embodiments, the immunogeniccarrier is selected from the group consisting of diphtheria toxoid,tetanus toxoid, keyhole limpet hemocyanin, and bovine serum albumin. Insome embodiments, the linker comprises a ε-maleimido caproic acidN-hydroxysuccinamide ester.

In some embodiments, the linker and the gastrin peptide are separated byan amino acid spacer, optionally wherein the amino acid spacer isbetween 1 and 10 amino acids in length, further optionally wherein theamino acid spacer is 7 amino acids in length.

In some embodiments, the presently disclosed pharmaceutical compositionsfurther comprise an adjuvant, optionally an oil-based adjuvant.

In some embodiments, the immune checkpoint inhibitor inhibits abiological activity of a target polypeptide selected from the groupconsisting of cytotoxic T-lymphocyte antigen 4 (CTLA4), programmed celldeath-1 receptor (PD-1), and programmed cell death 1 receptor ligand(PD-L1). In some embodiments, the immune checkpoint inhibitor isselected from the group consisting of Ipilimumab, Tremelimumab,Nivolumab, Pidilizumab, Pembrolizumab, AMP514, AUNP12,BMS-936559/MDX-1105, Atezolizumab, MPDL3280A, RG7446, R05541267,MEDI4736, Avelumab and Durvalumab.

In some embodiments, the gastrin-associated tumor and/or cancer ispancreatic cancer.

In some embodiments of the presently disclosed pharmaceuticalcompositions, the first agent comprises an amount of a gastrin peptidecomprising, consisting essentially of, or consisting of an amino acidsequence selected from the group consisting of EGPWLEEEEE (SEQ ID NO:1), EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY (SEQ ID NO: 3), andEGPWLEEEEEAYGWMDF (SEQ ID NO: 4) effective to induce an anti-gastrinhumoral response and the second agent comprises an amount of an immunecheckpoint inhibitor that is effective to induce or enhance a cellularimmune response against a gastrin-associated tumor or cancer whenadministered to a subject who has gastrin-associated tumor or cancer. Insome embodiments, the glutamic acid residue at amino acid 1 of any ofSEQ ID NOs: 1-4 is a pyroglutamate residue.

In some embodiments of the presently disclosed pharmaceuticalcompositions, the first agent comprises one or more anti-CCK-B receptorantibodies and is present in the pharmaceutical composition in an amountsufficient to reduce or inhibit gastrin signaling via CCK-B receptorspresent on a gastrin-associated tumor or cancer when administered to asubject that has a gastrin-associated tumor or cancer.

In some embodiments, the presently disclosed subject matter alsoprovides methods for treating a gastrin-associated tumor or cancer in asubject. In some embodiments, the methods comprise administering to thesubject an effective amount of a composition that comprises a firstagent that induces and/or provides an active and/or a passive humoralimmune response against a gastrin peptide and/or a CCK-B receptor; and asecond agent that induces and/or provides a cellular immune responseagainst the gastrin-associated tumor or cancer. In some embodiments, thefirst agent is selected from the group consisting of a gastrin peptide,an anti-gastrin antibody, and an anti-CCK-R antibody. In someembodiments, the first agent comprises a gastrin peptide, optionally agastrin peptide comprising, consisting essentially of, or consisting ofan amino acid sequence selected from the group consisting of EGPWLEEEEE(SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY (SEQ ID NO: 3),and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4). In some embodiments, the glutamicacid residue at amino acid 1 of any of SEQ ID NOs: 1-4 is apyroglutamate residue. In some embodiments, the gastrin peptide isconjugated to an immunogenic carrier, optionally via a linker, which insome embodiments can be selected from the group consisting of diphtheriatoxoid, tetanus toxoid, keyhole limpet hemocyanin, and bovine serumalbumin. In some embodiments, the linker comprises a ε-maleimido caproicacid N-hydroxysuccinamide ester. In some embodiments, the linker and thegastrin peptide are separated by an amino acid spacer, optionallywherein the amino acid spacer is between 1 and 10 amino acids in length,further optionally wherein the amino acid spacer is 7 amino acids inlength. In some embodiments, the composition further comprises anadjuvant, optionally an oil-based adjuvant. In some embodiments, theinducer of the cellular immune response against the gastrin-associatedtumor or cancer comprises an immune checkpoint inhibitor. In someembodiments, the immune checkpoint inhibitor inhibits a biologicalactivity of a target polypeptide selected from the group consisting ofcytotoxic T-lymphocyte antigen 4 (CTLA4), programmed cell death-1receptor (PD-1), and programmed cell death 1 receptor ligand (PD-L1). Insome embodiments, the immune checkpoint inhibitor is selected from thegroup consisting of Ipilimumab, Tremelimumab, Nivolumab, Pidilizumab,Pembrolizumab, AMP514, AUNP12, BMS-936559/MDX-1105, Atezolizumab,MPDL3280A, RG7446, R05541267, MEDI4736, Avelumab and Durvalumab.

In some embodiments of the presently disclosed methods, thegastrin-associated tumor and/or cancer is pancreatic cancer. In someembodiments, the composition induces a reduction in and/or prevents thedevelopment of fibrosis associated with the pancreatic cancer. In someembodiments, the composition is administered in a dose selected from thegroup consisting of about 50 μg to about 1000 μg, about 50 μg to about500 μg, about 100 μg to about 1000 μg, about 200 μg to about 1000 μg,and about 250 μg to about 500 μg, and optionally wherein the dose isrepeated once, twice, or three times, optionally wherein the second doseis administered 1 week after the first dose and the third dose, ifadministered, is administered 1 or 2 weeks after the second dose.

In some embodiments, the presently disclosed subject matter alsoprovides methods for treating gastrin-associated tumors and/or cancers.In some embodiments, the methods comprise administering to a subject inneed thereof a first agent that directly or indirectly inhibits one ormore biological activities of gastrin in the tumor and/or cancer and asecond agent comprising a stimulator of a cellular immune responseagainst the tumor and/or the cancer. In some embodiments, the firstagent directly or indirectly inhibits one or more biological activitiesof gastrin in the tumor and/or cancer by providing and/or inducing ahumoral immune response against a gastrin peptide, optionally whereinthe agent is selected from the group consisting of an anti-gastrinantibody and a gastrin peptide that induces production of neutralizinganti-gastrin antibodies in the subject; and/or comprises a nucleic acidthat inhibits expression of a gastrin gene product. In some embodiments,the anti-gastrin antibody is an antibody directed against an epitopepresent within gastrin-17 (G17). In some embodiments, the epitope ispresent within the amino acid sequence EGPWLEEEEE (SEQ ID NO: 1),EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY (SEQ ID NO: 3), orEGPWLEEEEEAYGWMDF (SEQ ID NO: 4). In some embodiments, the gastrinpeptide comprises an amino acid sequence selected from the groupconsisting of EGPWLEEEEE (SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2),EGPWLEEEEEAY (SEQ ID NO: 3), and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4). Insome embodiments, the glutamic acid residue at amino acid 1 of any ofSEQ ID NOs: 1-4 is a pyroglutamate residue. In some embodiments, thefirst agent comprises the gastrin peptide conjugated to an immunogeniccarrier, optionally an immunogenic carrier selected from the groupconsisting of diphtheria toxoid, tetanus toxoid, keyhole limpethemocyanin, and bovine serum albumin. In some embodiments, the gastrinpeptide is conjugated to the immunogenic carrier via a linker,optionally a linker that comprises a ε-maleimido caproic acidN-hydroxysuccinamide ester. In some embodiments, the linker and thegastrin peptide are separated by an amino acid spacer, optionallywherein the amino acid spacer is between 1 and 10 amino acids in length,further optionally wherein the amino acid spacer is 7 amino acids inlength. In some embodiments, the first agent further comprises anadjuvant, optionally an oil-based adjuvant. In some embodiments, thesecond agent is an immune checkpoint inhibitor. In some embodiments, theimmune checkpoint inhibitor inhibits a biological activity of a targetpolypeptide selected from the group consisting of cytotoxic T-lymphocyteantigen 4 (CTLA4), programmed cell death-1 receptor (PD-1), andprogrammed cell death 1 receptor ligand (PD-L1). In some embodiments,the immune checkpoint inhibitor is selected from the group consisting ofIpilimumab, Tremelimumab, Nivolumab, Pidilizumab, Pembrolizumab, AMP514,AUNP12, BMS-936559/MDX-1105, Atezolizumab, MPDL3280A, RG7446, R05541267,MEDI4736, and Avelumab.

In some embodiments of the presently disclosed method, thegastrin-associated tumor and/or cancer is pancreatic cancer. In someembodiments, the first agent induces a reduction in and/or prevents thedevelopment of fibrosis associated with the pancreatic cancer.

In some embodiments, the presently disclosed subject matter providesmethods for inhibiting growth of a gastrin-associated tumor and/orcancer in a subject. In some embodiments, the methods compriseadministering to the subject a composition that comprises a first agentcomprising a gastrin immunogen, one or more anti-gastrin antibodies, oneor more anti-CCK-B receptor antibodies, or any combination thereof; anda second agent comprising an immune checkpoint inhibitor. In someembodiments, the first agent is selected from the group consisting of agastrin peptide, an anti-gastrin antibody, and an anti-CCK-R antibody.In some embodiments, the first agent comprises a gastrin peptide,optionally a gastrin peptide comprising, consisting essentially of, orconsisting of an amino acid sequence selected from the group consistingof EGPWLEEEEE (SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY(SEQ ID NO: 3), and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4). In someembodiments, the glutamic acid residue at amino acid 1 of any of SEQ IDNOs: 1-4 is a pyroglutamate residue. In some embodiments, the gastrinpeptide is conjugated to an immunogenic carrier, optionally wherein theimmunogenic carrier is selected from the group consisting of diphtheriatoxoid, tetanus toxoid, keyhole limpet hemocyanin, and bovine serumalbumin. In some embodiments, the gastrin peptide is conjugated to animmunogenic carrier via a linker, optionally wherein the linkercomprises a ε-maleimido caproic acid N-hydroxysuccinamide ester. In someembodiments, the linker and the gastrin peptide are separated by anamino acid spacer, optionally wherein the amino acid spacer is between 1and 10 amino acids in length, further optionally wherein the amino acidspacer is 7 amino acids in length. In some embodiments, methods employ acomposition that further comprises an adjuvant, optionally an oil-basedadjuvant. In some embodiments, the inducer of the cellular immuneresponse against the gastrin-associated tumor or cancer comprises animmune checkpoint inhibitor. In some embodiments, the immune checkpointinhibitor inhibits a biological activity of a target polypeptideselected from the group consisting of cytotoxic T-lymphocyte antigen 4(CTLA4), programmed cell death-1 receptor (PD-1), and programmed celldeath 1 receptor ligand (PD-L1). In some embodiments, the immunecheckpoint inhibitor is selected from the group consisting ofIpilimumab, Tremelimumab, Nivolumab, Pidilizumab, Pembrolizumab, AMP514,AUNP12, BMS-936559/MDX-1105, Atezolizumab, MPDL3280A, RG7446, R05541267,MEDI4736, Avelumab and Durvalumab.

In some embodiments of the presently disclosed methods, thegastrin-associated tumor and/or cancer is pancreatic cancer. In someembodiments, the composition employed in the presently disclosed methodsinduces a reduction in and/or prevents the development of fibrosisassociated with the pancreatic cancer. In some embodiments, thecomposition is administered in a dose selected from the group consistingof about 50 μg to about 1000 μg, about 50 μg to about 500 μg, about 100μg to about 1000 μg, about 200 μg to about 1000 μg, and about 250 μg toabout 500 μg, and optionally wherein the dose is repeated once, twice,or three times, optionally wherein the second dose is administered 1week after the first dose and the third dose, if administered, isadministered 1 or 2 weeks after the second dose.

The presently disclosed subject matter also provides in some embodimentsmethods for inducing and/or enhancing cellular immune responses againstgastrin-associated tumors and/or cancers in subjects. In someembodiments, the methods comprise administering to a subject that has agastrin-associated tumor or cancer an effective amount of a compositioncomprising an agent that reduces or inhibits gastrin signaling via CCK-Breceptors present on a gastrin-associated tumor or cancer. In someembodiments, the agent comprises a gastrin peptide, an anti-gastrinantibody, an anti-CCK-B receptor antibody, or any combination thereof.In some embodiments, the gastrin peptide comprises a gastrin-17 (G17)peptide or an immunogenic fragment thereof. In some embodiments, thegastrin peptide or the immunogenic fragment thereof comprises, consistsessentially of, or consists of an amino acid sequence selected from thegroup consisting of EGPWLEEEEE (SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2),EGPWLEEEEEAY (SEQ ID NO: 3), and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4). Insome embodiments, the glutamic acid residue at amino acid 1 of any ofSEQ ID NOs: 1-4 is a pyroglutamate residue. In some embodiments, theagent comprises a gastrin peptide conjugated to an immunogenic carrier,optionally wherein the immunogenic carrier is selected from the groupconsisting of diphtheria toxoid, tetanus toxoid, keyhole limpethemocyanin, and bovine serum albumin. In some embodiments, the gastrinpeptide is conjugated to the immunogenic carrier via a linker,optionally a linker comprising a ε-maleimido caproic acidN-hydroxysuccinamide ester. In some embodiments, the linker and thegastrin peptide are separated by an amino acid spacer, optionallywherein the amino acid spacer is between 1 and 10 amino acids in length,further optionally wherein the amino acid spacer is 7 amino acids inlength. In some embodiments, the composition employed in the presentlydisclosed methods further comprises an adjuvant, optionally an oil-basedadjuvant.

In some embodiments, the presently disclosed subject matter alsoprovides methods for sensitizing tumors and/or cancers associated withgastrin and/or CCK-B receptor signaling in subject to inducers ofcellular immune responses directed against the tumors and/or cancers. Insome embodiments, the methods comprise administering to the subject acomposition comprising a first agent that induces and/or provides anactive and/or a passive humoral immune response against a gastrinpeptide, and a second agent that induces and/or provides a cellularimmune response against the tumor and/or the cancer, or a combinationthereof, optionally wherein the first agent and the second agent areindividually selected from the group consisting of a gastrin peptideand/or a fragment and/or a derivative thereof that induces a cellularimmune response or production of neutralizing anti-gastrin antibodies inthe subject and a neutralizing anti-gastrin antibody and/or a fragmentand/or derivative thereof and; and/or a composition comprising a nucleicacid that inhibits expression of a gastrin gene product; and/or acomposition comprising an agent that blocks the biological function ofgastrin at the CCK-B receptor. In some embodiments, the anti-gastrinantibody is an antibody directed against an epitope present withingastrin-17 (G17). In some embodiments, the epitope is present within theamino acid sequence EGPWLEEEEE (SEQ ID NO: 1) or EGPWLEEEE (SEQ ID NO:2) or EGPWLEEEEEAY (SEQ ID NO: 3) OR EGPWLEEEEEAYGWMDF (SEQ ID NO: 4).In some embodiments, the glutamic acid residue at amino acid 1 of any ofSEQ ID NOs: 1-4 is a pyroglutamate residue. In some embodiments, thecomposition comprises the gastrin peptide conjugated to an immunogeniccarrier. In some embodiments, the gastrin peptide comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. In some embodiments, theimmunogenic carrier is selected from the group consisting of diphtheriatoxoid, tetanus toxoid, keyhole limpet hemocyanin, and bovine serumalbumin. In some embodiments, the gastrin peptide is conjugated to theimmunogenic carrier via a linker. In some embodiments, the linkercomprises a ε-maleimido caproic acid N-hydroxysuccinamide ester. In someembodiments, the linker and the gastrin peptide are separated by anamino acid spacer, optionally wherein the amino acid spacer is between 1and 10 amino acids in length, further optionally wherein the amino acidspacer is 7 amino acids in length. In some embodiments, the compositionemployed in the presently disclosed methods further comprises anadjuvant, optionally an oil-based adjuvant. In some embodiments, theinducer of the cellular immune response against the gastrin-associatedtumor and/or cancer comprises an immune checkpoint inhibitor. In someembodiments, the immune checkpoint inhibitor inhibits a biologicalactivity of a target polypeptide selected from the group consisting ofcytotoxic T-lymphocyte antigen 4 (CTLA4), programmed cell death-1receptor (PD-1), and programmed cell death 1 receptor ligand (PD-L1). Insome embodiments, the immune checkpoint inhibitor is selected from thegroup consisting of Ipilimumab, Tremelimumab, Nivolumab, Pidilizumab,Pembrolizumab, AMP514, AUNP12, BMS-936559/MDX-1105, Atezolizumab,MPDL3280A, RG7446, R05541267, MEDI4736, Avelumab and Durvalumab.

In some embodiments of the presently disclosed methods, thegastrin-associated tumor and/or cancer is pancreatic cancer. In someembodiments, the composition induces a reduction in and/or prevents thedevelopment of fibrosis associated with the pancreatic cancer. In someembodiments, the composition is administered in a dose selected from thegroup consisting of about 50 μg to about 1000 μg, about 50 μg to about500 μg, about 100 μg to about 1000 μg, about 200 μg to about 1000 μg,and about 250 μg to about 500 μg, and optionally wherein the dose isrepeated once, twice, or three times, optionally wherein the second doseis administered 1 week after the first dose and the third dose, ifadministered, is administered 1 or 2 weeks after the second dose.

The presently disclosed subject matter also provides in some embodimentsmethods for preventing, reducing, and/or eliminating formation offibrosis associated with tumors and/or cancers. In some embodiments, themethods comprise contacting cells of tumors and/or cancers with an agentthat directly or indirectly inhibits one or more biological activitiesof gastrin in the tumor and/or cancer. In some embodiments, the agentprovides and/or induces a humoral immune response against a gastrinpeptide, optionally wherein the agent is selected from the groupconsisting of an anti-gastrin antibody, and/or a fragment and/orderivative thereof, and a gastrin peptide that induces production ofneutralizing anti-gastrin antibodies in the subject; and/or comprises anucleic acid that inhibits expression of a gastrin gene product; and/orcomprises a small molecule compound that blocks the function of thegastrin hormone. In some embodiments, the anti-gastrin antibody is anantibody directed against an epitope present within gastrin-17 (G17). Insome embodiments, the epitope is present within the amino acid sequenceEGPWLEEEEE (SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY (SEQID NO: 3), or EGPWLEEEEEAYGWMDF (SEQ ID NO: 4). In some embodiments, theglutamic acid residue at amino acid 1 of any of SEQ ID NOs: 1-4 is apyroglutamate residue. In some embodiments, the agent comprises thegastrin peptide that induces production of neutralizing anti-gastrinantibodies conjugated to an immunogenic carrier. In some embodiments,the gastrin peptide comprises an amino acid sequence selected from thegroup consisting of EGPWLEEEEE (SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2),EGPWLEEEEEAY (SEQ ID NO: 3), and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4). Insome embodiments, the glutamic acid residue at amino acid 1 of any ofSEQ ID NOs: 1-4 is a pyroglutamate residue. In some embodiments, theimmunogenic carrier is selected from the group consisting of diphtheriatoxoid, tetanus toxoid, keyhole limpet hemocyanin, and bovine serumalbumin. In some embodiments, the gastrin peptide is conjugated to theimmunogenic carrier via a linker, optionally a linker comprising aε-maleimido caproic acid N-hydroxysuccinamide ester. In someembodiments, the linker and the gastrin peptide are separated by anamino acid spacer, optionally wherein the amino acid spacer is between 1and 10 amino acids in length, further optionally wherein the amino acidspacer is 7 amino acids in length. In some embodiments, the agentfurther comprises an adjuvant, optionally an oil-based adjuvant. In someembodiments, the presently disclosed methods further comprise contactingthe tumor and/or the cancer with a second agent comprising a stimulatorof a cellular immune response against the tumor and/or the cancer. Insome embodiments, the second agent is an immune checkpoint inhibitor.Isae, the immune checkpoint inhibitor inhibits a biological activity ofa target polypeptide selected from the group consisting of cytotoxicT-lymphocyte antigen 4 (CTLA4), programmed cell death-1 receptor (PD-1),and programmed cell death 1 receptor ligand (PD-L1). In someembodiments, the immune checkpoint inhibitor is selected from the groupconsisting of Ipilimumab, Tremelimumab, Nivolumab, Pidilizumab,Pembrolizumab, AMP514, AUNP12, BMS-936559/MDX-1105, Atezolizumab,MPDL3280A, RG7446, R05541267, MEDI4736, and Avelumab. In someembodiments of the presently disclosed methods, the tumor and/or canceris pancreatic cancer.

The presently disclosed subject matter also provides in some embodimentsuses of the pharmaceutical compositions disclosed herein for producing amedicament for treating a gastrin-associated tumor or cancer.

The presently disclosed subject matter also provides in some embodimentsuses of the presently disclosed pharmaceutical compositions for treatinga gastrin-associated tumors and/or cancer.

The presently disclosed subject matter also provides in some embodimentsuses of the presently disclosed pharmaceutical compositions forpreventing, reducing, and/or eliminating metastasis of agastrin-associated tumor or cancer by administering to a subject havinga gastrin-associated tumor or cancer an amount of a pharmaceuticalcomposition as disclosed herein sufficient to enhance the number ofCD4⁻/CD8⁻ T_(EMRA) cells in the subject that respond to thegastrin-associated tumor or cancer.

The presently disclosed subject matter also provides in some embodimentsuses of the presently disclosed pharmaceutical compositions forincreasing the number of T_(EMRA) cells in a subject that respond to agastrin-associated tumor or cancer.

The presently disclosed subject matter also provides in some embodimentsuses of compositions comprising an immune checkpoint inhibitor and agastrin immunogen to treat a gastrin-associated tumor or cancer.

The presently disclosed subject matter also provides in some embodimentsuses of compositions comprising an immune checkpoint inhibitor and agastrin immunogen for the preparation of a medicament to treat agastrin-associated tumor or cancer.

The presently disclosed subject matter also provides in some embodimentsuses of the presently disclosed pharmaceutical compositions forpreventing, reducing, and/or eliminating metastasis of agastrin-associated tumor or cancer. In some embodiments, the presentlydisclosed subject matter relates to administering to a subject having agastrin-associated tumor or cancer an amount of a pharmaceuticalcomposition as disclosed herein sufficient to enhance the number of CD8⁺tumor infiltrating lymphocytes. In some embodiments, the use results inimproved survival, reduction of tumor growth, and/or enhanced efficacyof chemotherapy and/or immune checkpoint therapy as compared to thatseen in subjects that do not receive a pharmaceutical composition asdisclosed herein.

Use of the pharmaceutical composition of any one of claims 1-13 forpreventing, reducing, and/or eliminating metastasis of agastrin-associated tumor or cancer by administering to a subject havinga gastrin-associated tumor or cancer an amount of the pharmaceuticalcomposition of any one of claims 1-13 sufficient to reduce the number ofFoxP3⁺ inhibitory T-regulatory cells.

Thus, it is an object of the presently disclosed subject matter toprovide a method for treating gastrin-associated or CCK-Breceptor-containing tumors and/or cancers.

An object of the presently disclosed subject matter having been statedhereinabove, and which is achieved in whole or in part by thecompositions and methods disclosed herein, other objects will becomeevident as the description proceeds when taken in connection with theaccompanying Figures as best described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an exemplary experimental strategy for testingthe ability of patent applications with or without an immune checkpointinhibitor to influence growth of a pancreatic cell tumor in mice. In aparticular embodiment, subcutaneous tumors were produced by injectingC57BL/6 mice with 5×10⁵ murine mT3 pancreatic cancer cells (C57BL/6 issyngeneic with mT3 cells) at −1 week before treatment. Groups of 10 mice(40 total) were treated with PAS at t=0, 1, and 3 weeks and/or ananti-PD-1 antibody (PD1-1 Ab; Bio X cell, West Lebanon, N.H., UnitedStates of America) at t=0, 4, 8, 15, and 21 days. Between treatments,tumor volumes were measured. The study was ended and PBMC were collectedfrom spleens and tumors were excised from the mice and analyzed.

FIG. 2 is a bar graph showing mean tumor weights in grams in mT3-bearingmice following treatment with control (phosphate-buffered saline only;PBS), PAS alone (100 μg per administration; PAS100), PD-1 Ab alone (150μg per administration; PD-1), or the combination of PAS (100 μg peradministration) and PD-1 Ab (150 μg per administration; PAS100+PD-1).NS: p 0.05 (i.e., not significant); * p<0.05 compared to PBS andcompared to PAS100; # p=0.0017 as compared to PD-1. Error bars are ±SEM.

FIGS. 3A and 3B are a series of plots of CD4⁻/CD8⁻ and CD4⁻/CD8⁻T_(EMRA) cells present in CD3 terminally differentiated T cells aftertreatment with PBS, PD-1 Ab alone (150 μg per administration; PD1), PASalone (100 μg per administration; PAS), or the combination of PAS (100μg per administration) and PD-1 Ab (150 μg per administration; PAS/PD1).FIG. 3A shows the percentages of CD3⁺/CD4⁻/CD8⁻ andCD3⁺/CD4⁻/CD8⁻/CD44⁻/CD62L⁻ (i.e. T_(EMRA)) cells in mice that underwentvarious treatments. FIG. 3B shows the portion of CD3⁺/CD4⁻/CD8⁻ cellsthat were CD3⁺/CD4⁻/CD8⁻/CD44⁻/CD62L⁻ T_(EMRA) cells. The portion ofcells was calculated by taking the percentage of CD3⁺/CD4⁻/CD8⁻lymphocytes multiplied by the percentage of CD3⁺/CD4⁻/CD8⁻/CD44⁻/CD62L⁻T_(EMRA) cells in the CD4⁻/CD8⁻ lymphocytes/10000 to calculate theportion of CD4⁻/CD8⁻/CD44⁻/CD62L⁻ T_(EMRA) cells in the CD3⁺ T cellfraction (see FIG. 3B). * p<0.05; ** p<0.01. Error bars are ±1 standarddeviation.

FIGS. 4A and 4B are a series of bar graphs summarizing a cytokineactivation assay with respect to TNFα, Granzyme B, Perforin, and INFγ invarious T cell subpopulations without (FIG. 4A) and with (FIG. 4B)re-stimulation with gastrin after treatment with PAS100. FIG. 4A showsthat the T cells isolated from the spleens of mice treated with PAS100were indeed activated. When these same cells were re-stimulated withgastrin in culture for 6 hours (FIG. 4B), they were re-stimulated andreleased even more of each cytokine. Black bars: INFγ. Light gray bars:Granzyme B. Dark gray bars: Perforin. Hatched gray bars: TNFα.

FIGS. 5A and 5B are a series of bar graphs comparing cytokine releasewith respect to TNFα, Granzyme B, Perforin, and INFγ in CD4⁻/CD8⁻ (leftgroup in each Figure), CD8⁺ (middle group in each Figure), and CD4⁺(right group in each Figure) T cell subpopulations treated with PAS100monotherapy (FIG. 5A) or PAS100+PD-1 combination therapy (FIG. 5B).Activated T lymphocytes released increased cytokines compared tolymphocytes from PBS treated mice. The lymphocytes from the combinationtreated mice released markedly higher levels of cytokines, suggestingthat the combination therapy was better at stimulating activated Tcells. TNFα in particular was increased greater than 2-fold with thePAS+PD-1 Ab combination therapy. Black bars: INFγ. Light gray bars:Granzyme B. Dark gray bars: Perforin. Hatched gray bars: TNFα.

FIGS. 6A and 6B show the results of PBS control, PD-1 monotherapy,PAS100 monotherapy, and PAS100 & PD-1 combination therapy on thedevelopment of fibrosis in mT3 pancreatic cancer cell tumors in mice.FIG. 6A depicts mT3 tumors stained with Masson's Trichrome Stain, whichstains collagen blue and provides an indicator of fibrosis. FIG. 6B is abar graph summarizing the results of the staining depicted in FIG. 6A.Of note is that whereas the integrated density of the tumors treatedwith PD-1 monotherapy and PAS100 monotherapy were insignificantlydifferent the negative control PBS treatment, the PAS+PD-1 Abcombination therapy resulted in a decrease in density (and hencefibrosis) that was statistically significant as compared to PBS alone(p<0.005) and also PAS100 alone (p<0.001). ***p<0.005 compared to PBSand p<0.001 as compared to PAS100. Black bar: PBS. Light gray bar: PD-1alone. White bar: PAS100 alone. Hatched gray bar: PAS100+PD-1.

FIGS. 7A and 7B show the results of PBS control, PD-1 monotherapy,PAS100 monotherapy, and PAS100 & PD-1 combination therapy oninfiltration of CD8⁺ cells into mT3 pancreatic cancer cell tumors inmice. FIG. 7A depicts exemplary mT3 tumors stained with an antibody thatbinds to the CD8 after treatment with PBS, PD-1 Ab (PD-1) monotherapy,PAS100 monotherapy (patent applications), and PAS100 & PD-1 combinationtherapy on infiltration of CD8⁺ cells into mT3 pancreatic cancer celltumors in mice. FIG. 7B is a bar graph summarizing the data exemplifiedby FIG. 7A. Treatment with PD-1 (PD-1 Ab) monotherapy or PAS100 aloneresulted in significantly higher levels of CD8⁺ cells in tumors(p=0.0019 and p=0.0026, respectively) as compared to the negativecontrol PBS treatment. The PAS+PD-1 Ab combination therapy resulted ineven greater levels of CD8⁺ cells in tumors when compared to PBS alone(p=4.7×10⁻⁵) as well as when compared to PD-1 alone (p=0.042) and whencompared to PAS100 alone (p=0.039). PD-1 alone compared to PAS100 alonewas not significantly different (p>0.05). **p=0.0026; ***p=0.0019;****p=4.7×10⁻⁵ as compared to PBS. Error bars are ±SEM.

FIGS. 8A and 8B depict analyses of Foxp3⁺ cells in mT3 tumors. FIG. 8Adepicts exemplary mT3 tumors stained with an antibody that binds to theFoxp3 protein, a marker for T_(regs). Comparison of the fields showsthat as compared to PBS (upper left panel), PD-1 monotherapy (upperright panel), or PAS100 monotherapy (lower left panel), PAS100 & PD-1combination therapy resulted in a decrease in the presence ofintratumoral T_(regs), suggesting that PAS100+PD-1 combination therapymight modify the intratumoral environment to an extent where theintratumoral microenvironment might be characterized by a lower degreeof T_(reg)-based immunosuppression as compared to either monotherapyalone. FIG. 8B is a bar graph summarizing the data exemplified by FIG.8A. As compared to PBS, the number of Foxp3⁺ cells in tumors treatedwith PD-1 monotherapy or PAS100 monotherapy was not significantlydifferent. Tumors treated with PAS100+PD-1 Ab combination therapy hadsignificantly fewer Foxp3⁺ cells than the negative control. * p=0.038.Black bar: PBS. Light gray bar: PD-1 Ab alone. White bar: PAS100 alone.Hatched gray bar: PAS100+PD-1 Ab. Error bars are ±SEM.

DETAILED DESCRIPTION

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts can haveapplicability in other sections throughout the entire description.

I. GENERAL CONSIDERATIONS

The presently disclosed subject matter relates in some embodiments tomethods and systems for treating human and animal cancers usingcombinations of treatments that together generate both a humoral immuneanti-tumor effect plus a cellular immune anti-tumor effect. Moreparticularly, the presently disclosed subject matter relates in someembodiments to using particular combinations of drugs that: (1) induceimmunologic humoral B cell responses that generate antibodies againstthe tumor and/or circulating tumor growth factor(s); and (2) induce orotherwise enhance immunologic cellular T cell responses directed againstthe tumor to elicit cytotoxic T lymphocyte responses. More particularly,the presently disclosed subject matter relates in some embodiments tomethods and systems for treating human cancers using an anti-gastrincancer vaccine in combination with a second drug that causes immunecheckpoint blockade. Even more particularly, the presently disclosedsubject matter relates in some embodiments to treating specific humancancers with one or more cancer vaccines designed to elicit a B cellantibody response to the active form of the growth factor gastrin. Asdisclosed herein for the first time, in some embodiments anti-gastrinvaccines can result in a human tumor becoming responsive to treatmentwith an immune checkpoint inhibitor, thus creating an unexpectedadditive or even synergistic combination therapy effect that enhancesoverall anti-tumor efficacy.

The presently disclosed subject matter also relates in some embodimentsto methods for the treatment of tumors and/or cancers using acombination of methods, which generate both a humoral antibody immuneresponse (a gastrin cancer vaccine) and a cellular T cell immuneresponse (immune checkpoint blockade). In some embodiments, thepresently disclosed subject matter relates to compositions and methodsthat produce novel, unexpected, additive, and/or synergistic efficacy intreating human and animal gastrointestinal tumors using a novel andunique combination of drug classes which generate both a humoral immuneanti-tumor effect plus a cellular immune anti-tumor effect. In someembodiments, the presently disclosed subject matter relates to usingspecific combinations of drugs that: (1) induce immunologic humoral Bcell responses to tumor growth factors and/or circulating tumor growthfactors; and (2) cause and/or enhance immunologic cellular T cellresponses directed against tumors to elicit cytotoxic T lymphocyteresponses. In some embodiments, the presently disclosed subject matterrelates to methods and systems for treating human and animal cancersusing the presently disclosed combinations of gastrin cancer vaccinesand one or more second drugs that causes immune checkpoint blockade. Insome embodiments, the presently disclosed subject matter relates totreating specific human cancers with one or more cancer vaccinesdesigned to elicit B cell antibody responses to the active form of thegrowth factor gastrin, which as disclosed herein unexpectedly alsoresults in the human tumor becoming more responsive to the treatmentwith an immune checkpoint inhibitor, thus creating an unexpected,additive, or even synergistic combination therapy effect that enhancesanti-tumor efficacy. In some embodiments, the presently disclosedsubject matter thus relates to using PAS with immune checkpointinhibitors. In some embodiments, the presently disclosed subject matterrelates to using PAS as a cancer vaccine to induce both a humoral and acellular immune response.

II. DEFINITIONS

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the presentlydisclosed subject matter.

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. References to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques that would be apparent to one of skill in the art.While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

In describing the presently disclosed subject matter, it will beunderstood that a number of techniques and steps are disclosed. Each ofthese has individual benefit and each can also be used in conjunctionwith one or more, or in some cases all, of the other disclosedtechniques.

Accordingly, for the sake of clarity, this description will refrain fromrepeating every possible combination of the individual steps in anunnecessary fashion. Nevertheless, the specification and claims shouldbe read with the understanding that such combinations are entirelywithin the scope of the presently disclosed and claimed subject matter.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, including inthe claims. For example, the phrase “an inhibitor” refers to one or moreinhibitors, including a plurality of the same inhibitor. Similarly, thephrase “at least one”, when employed herein to refer to an entity,refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 75, 100, or more of that entity, including but notlimited to whole number values between 1 and 100 and greater than 100.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about”. The term “about”, as used herein when referring to ameasurable value such as an amount of mass, weight, time, volume,concentration, or percentage, is meant to encompass variations of insome embodiments ±20%, in some embodiments ±10%, in some embodiments±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in someembodiments ±0.1% from the specified amount, as such variations areappropriate to perform the disclosed methods and/or employ the disclosedcompositions. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that can vary depending upon the desired propertiessought to be obtained by the presently disclosed subject matter.

As used herein, the term “and/or” when used in the context of a list ofentities, refers to the entities being present singly or in combination.Thus, for example, the phrase “A, B, C, and/or D” includes A, B, C, andD individually, but also includes any and all combinations andsubcombinations of A, B, C, and D.

As used herein, the terms “antibody” and “antibodies” refer to proteinscomprising one or more polypeptides substantially encoded byimmunoglobulin genes or fragments of immunoglobulin genes.Immunoglobulin genes typically include the kappa (κ), lambda (λ), alpha(α), gamma (γ), delta (δ), epsilon (ε), and mu (μ) constant regiongenes, as well as myriad immunoglobulin variable region genes. Lightchains are classified as either κ or λ. In mammals, heavy chains areclassified as γ, μ, α, δ, or ε, which in turn define the immunoglobulinclasses, IgG, IgM, IgA, IgD, and IgE, respectively. Other species haveother light and heavy chain genes (e.g., certain avians produced what isreferred to as IgY, which is an immunoglobulin type that hens deposit inthe yolks of their eggs), which are similarly encompassed by thepresently disclosed subject matter. In some embodiments, the term“antibody” refers to an antibody that binds specifically to an epitopethat is present on a gastrin gene product, including but not limited toan epitope that is present within an amino acid sequence as set forth inSEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” chain (averagemolecular weight of about 25 kilodalton (kDa)) and one “heavy” chain(average molecular weight of about 50-70 kDa). The two identical pairsof polypeptide chains are held together in dimeric form by disulfidebonds that are present within the heavy chain region. The N-terminus ofeach chain defines a variable region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. The terms variablelight chain (V_(L)) and variable heavy chain (V_(H)) refer to theselight and heavy chains, respectively.

Antibodies typically exist as intact immunoglobulins or as a number ofwell-characterized fragments that can be produced by digestion withvarious peptidases. For example, digestion of an antibody molecule withpapain cleaves the antibody at a position N-terminal to the disulfidebonds. This produces three fragments: two identical “Fab” fragments,which have a light chain and the N-terminus of the heavy chain, and an“Fc” fragment that includes the C-terminus of the heavy chains heldtogether by the disulfide bonds. Pepsin, on the other hand, digests anantibody C-terminal to the disulfide bond in the hinge region to producea fragment known as the “F(ab)′₂” fragment, which is a dimer of the Fabfragments joined by the disulfide bond. The F(ab)′₂ fragment can bereduced under mild conditions to break the disulfide linkage in thehinge region, thereby converting the F(ab′)₂ dimer into two Fab′monomers. The Fab′ monomer is essentially an Fab fragment with part ofthe hinge region (see e.g., Paul, 1993 for a more detailed descriptionof other antibody fragments). With respect to these various fragments,Fab, F(ab′)₂, and Fab′ fragments include at least one intact antigenbinding domain, and thus are capable of binding to antigens.

While various antibody fragments are defined in terms of the digestionof an intact antibody, one of skill will appreciate that various ofthese fragments (including, but not limited to Fab′ fragments) can besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term “antibody” as used herein also includesantibody fragments either produced by the modification of wholeantibodies or synthesized de novo using recombinant DNA methodologies.In some embodiments, the term “antibody” comprises a fragment that hasat least one antigen binding domain.

Antibodies can be polyclonal or monoclonal. As used herein, the term“polyclonal” refers to antibodies that are derived from differentantibody-producing cells (e.g., B cells) that are present together in agiven collection of antibodies. Exemplary polyclonal antibodies includebut are not limited to those antibodies that bind to a particularantigen and that are found in the blood of an animal after that animalhas produced an immune response against the antigen. However, it isunderstood that a polyclonal preparation of antibodies can also beprepared artificially by mixing at least non-identical two antibodies.Thus, polyclonal antibodies typically include different antibodies thatare directed against (i.e., binds to) different epitopes (sometimesreferred to as an “antigenic determinant” or just “determinant”) of anygiven antigen.

As used herein, the term “monoclonal” refers to a single antibodyspecies and/or a substantially homogeneous population of a singleantibody species. Stated another way, “monoclonal” refers to individualantibodies or populations of individual antibodies in which theantibodies are identical in specificity and affinity except for possiblenaturally occurring mutations, or post-translational modifications thatcan be present in minor amounts. Typically, a monoclonal antibody (mAb)is generated by a single B cell or a progeny cell thereof (although thepresently disclosed subject matter also encompasses “monoclonal”antibodies that are produced by molecular biological techniques asdescribed herein). Monoclonal antibodies (mAbs) are highly specific,typically being directed against a single antigenic site. Furthermore,in contrast to polyclonal antibody preparations, a given mAb istypically directed against a single epitope on the antigen.

In addition to their specificity, mAbs can be advantageous for somepurposes in that they can be synthesized uncontaminated by otherantibodies. The modifier “monoclonal” is not to be construed asrequiring production of the antibody by any particular method, however.For example, in some embodiments, the mAbs of the presently disclosedsubject matter are prepared using the hybridoma methodology firstdescribed by Kohler et al., 1975, and in some embodiments, are madeusing recombinant DNA methods in bacterial or eukaryotic animal or plantcells (see e.g., U.S. Pat. No. 4,816,567, the entire contents of whichare incorporated herein by reference). mAbs can also be isolated fromphage antibody libraries using the techniques described in Clackson etal., 1991 and Marks et al., 1991, for example.

The antibodies, fragments, and derivatives of the presently disclosedsubject matter can also include chimeric antibodies. As used herein inthe context of antibodies, the term “chimeric”, and grammatical variantsthereof, refers to antibody derivatives that have constant regionsderived substantially or exclusively from antibody constant regions fromone species and variable regions derived substantially or exclusivelyfrom the sequence of the variable region from another species. Aparticular kind of chimeric antibody is a “humanized” antibody, in whichthe antibodies are produced by substituting the complementaritydetermining regions (CDRs) of, for example, a mouse antibody, for theCDRs of a human antibody (see e.g., PCT International Patent ApplicationPublication No. WO 1992/22653). Thus, in some embodiments, a humanizedantibody has constant regions and variable regions other than the CDRsthat are derived substantially or exclusively from the correspondinghuman antibody regions, and CDRs that are derived substantially orexclusively from a mammal other than a human.

The antibodies, fragments, and derivatives of the presently disclosedsubject matter can also be single chain antibodies and single chainantibody fragments. Single-chain antibody fragments contain amino acidsequences having at least one of the variable regions and/or CDRs of thewhole antibodies described herein but are lacking some or all of theconstant domains of those antibodies. These constant domains are notnecessary for antigen binding but constitute a major portion of thestructure of whole antibodies.

Single-chain antibody fragments can overcome some of the problemsassociated with the use of antibodies containing a part or all of aconstant domain. For example, single-chain antibody fragments tend to befree of undesired interactions between biological molecules and theheavy-chain constant region, or other unwanted biological activity.Additionally, single-chain antibody fragments are considerably smallerthan whole antibodies and can therefore have greater capillarypermeability than whole antibodies, allowing single-chain antibodyfragments to localize and bind to target antigen-binding sites moreefficiently. Also, antibody fragments can be produced on a relativelylarge scale in prokaryotic cells, thus facilitating their production.Furthermore, the relatively small size of single-chain antibodyfragments makes them less likely to provoke an immune response in arecipient than whole antibodies. The single-chain antibody fragments ofthe presently disclosed subject matter include but are not limited tosingle chain fragment variable (scFv) antibodies and derivatives thereofsuch as, but not limited to tandem di-scFv, tandem tri-scFv, diabodies,and triabodies, tetrabodies, miniantibodies, and minibodies.

Fv fragments correspond to the variable fragments at the N-termini ofimmunoglobulin heavy and light chains. Fv fragments appear to have lowerinteraction energy of their two chains than Fab fragments. To stabilizethe association of the V_(H) and V_(L) domains, they have been linkedwith peptides (see Bird et al., 1988; Huston et al., 1988), disulfidebridges (Glockshuber et al., 1990), and “knob in hole” mutations (Zhu etal., 1997). ScFv fragments can be produced by methods well known tothose skilled in the art (see e.g., Whitlow et al., 1991 and Huston etal., 1993.

scFv can be produced in bacterial cells such as E. coli or in eukaryoticcells. One potential disadvantage of scFv is the monovalency of theproduct, which can preclude an increased avidity due to polyvalentbinding, and their short half-life. Attempts to overcome these problemsinclude bivalent (scFv′)₂ produced from scFv containing an additionalC-terminal cysteine by chemical coupling (Adams et al., 1993; McCartneyet al., 1995) or by spontaneous site-specific dimerization of scFvcontaining an unpaired C-terminal cysteine residue (see Kipriyanov etal., 1995).

Alternatively, scFv can be forced to form multimers by shortening thepeptide linker to 3 to 12 residues to form “diabodies” (see Holliger etal., 1993). Reducing the linker still further can result in scFv trimers(“triabodies”; see Kortt et al., 1997) and tetramers (“tetrabodies”; seeLe Gall et al., 1999). Construction of bivalent scFv molecules can alsobe achieved by genetic fusion with protein dimerizing motifs to form“miniantibodies” (see Pack et al., 1992) and “minibodies” (see Hu etal., 1996). scFv-scFv tandems ((scFv)₂) can be produced by linking twoscFv units by a third peptide linker (see Kurucz et al., 1995).

Bispecific diabodies can be produced through the non-covalentassociation of two single chain fusion products consisting of V_(H)domain from one antibody connected by a short linker to the V_(L) domainof another antibody (see Kipriyanov et al., 1998). The stability of suchbispecific diabodies can be enhanced by the introduction of disulfidebridges or “knob in hole” mutations as described hereinabove or by theformation of single chain diabodies (scDb) wherein two hybrid scFvfragments are connected through a peptide linker (see Kontermann et al.,1999).

Tetravalent bispecific molecules can be produced, for example, by fusingan scFv fragment to the CH₃ domain of an IgG molecule or to a Fabfragment through the hinge region (see Coloma et al., 1997).Alternatively, tetravalent bispecific molecules have been created by thefusion of bispecific single chain diabodies (see Alt et al., 1999).Smaller tetravalent bispecific molecules can also be formed by thedimerization of either scFv-scFv tandems with a linker containing ahelix-loop-helix motif (DiBi miniantibodies; see Muller et al., 1998) ora single chain molecule comprising four antibody variable domains (V_(H)and V_(L)) in an orientation preventing intramolecular pairing (tandemdiabody; see Kipriyanov et al., 1999).

Bispecific F(ab′)₂ fragments can be created by chemical coupling of Fab′fragments or by heterodimerization through leucine zippers (see Shalabyet al., 1992; Kostelny et al., 1992). Also available are isolated V_(H)and V_(L) domains (see U.S. Pat. Nos. 6,172,197; 6,248,516; and6,291,158).

The presently disclosed subject matter also includes functionalequivalents of anti-gastrin antibodies. As used herein, the phrase“functional equivalent” as it refers to an antibody refers to a moleculethat has binding characteristics that are comparable to those of a givenantibody. In some embodiments, chimerized, humanized, and single chainantibodies, as well as fragments thereof, are considered functionalequivalents of the corresponding antibodies upon which they are based.

Functional equivalents also include polypeptides with amino acidsequences substantially the same as the amino acid sequence of thevariable or hypervariable regions of the antibodies of the presentlydisclosed subject matter. As used herein with respect to amino acidsequences, the phrase “substantially the same” refers to a sequencewith, in some embodiments at least 80%, in some embodiments at least85%, in some embodiments at least about 90%, in some embodiments atleast 91%, in some embodiments at least 92%, in some embodiments atleast 93%, in some embodiments at least 94%, in some embodiments atleast 95%, in some embodiments at least 96%, in some embodiments atleast 97%, in some embodiments at least 98%, and in some embodiments atleast about 99% sequence identity to another amino acid sequence, asdetermined by the FASTA search method in accordance with Pearson &Lipman, 1988. In some embodiments, the percent identity calculation isperformed over the full length of the amino acid sequence of an antibodyof the presently disclosed subject matter.

Functional equivalents further include fragments of antibodies that havethe same or comparable binding characteristics to those of a wholeantibody of the presently disclosed subject matter. Such fragments cancontain one or both Fab fragments, the F(ab′)₂ fragment, the F(ab′)fragment, an Fv fragment, or any other fragment that includes at leastone antigen binding domain. In some embodiments, the antibody fragmentscontain all six CDRs of a whole antibody of the presently disclosedsubject matter, although fragments containing fewer than all of suchregions, such as three, four, or five CDRs, can also be functionalequivalents as defined herein. Further, functional equivalents can be orcan combine members of any one of the following immunoglobulin classes:IgG, IgM, IgA, IgD, and IgE, and the subclasses thereof, as well asother subclasses as might be appropriate for non-mammalian subjects(e.g., IgY for chickens and other avian species).

Functional equivalents further include peptides that have the same orcomparable characteristics to those of a whole protein of the presentlydisclosed subject matter. Such peptides can contain one or more antigensof the whole protein, which can elicit an immune response in the treatedsubject.

Functional equivalents also include aptamers and other non-antibodymolecules, provided that such molecules have the same or comparablebinding characteristics to those of a whole antibody of the presentlydisclosed subject matter.

The term “comprising”, which is synonymous with “including”“containing”, or “characterized by”, is inclusive or open-ended and doesnot exclude additional, unrecited elements and/or method steps.“Comprising” is a term of art that means that the named elements and/orsteps are present, but that other elements and/or steps can be added andstill fall within the scope of the relevant subject matter.

As used herein, the phrase “consisting of” excludes any element, step,or ingredient not specifically recited. It is noted that, when thephrase “consists of” appears in a clause of the body of a claim, ratherthan immediately following the preamble, it limits only the element setforth in that clause; other elements are not excluded from the claim asa whole.

As used herein, the phrase “consisting essentially of” limits the scopeof the related disclosure or claim to the specified materials and/orsteps, plus those that do not materially affect the basic and novelcharacteristic(s) of the disclosed and/or claimed subject matter. Forexample, a pharmaceutical composition can “consist essentially of” apharmaceutically active agent or a plurality of pharmaceutically activeagents, which means that the recited pharmaceutically active agent(s)is/are the only pharmaceutically active agent(s) present in thepharmaceutical composition. It is noted, however, that carriers,excipients, and/or other inactive agents can and likely would be presentin such a pharmaceutical composition and are encompassed within thenature of the phrase “consisting essentially of”.

With respect to the terms “comprising”, “consisting of”, and “consistingessentially of”, where one of these three terms is used herein, thepresently disclosed and claimed subject matter can include the use ofeither of the other two terms. For example, in some embodiments, thepresently disclosed subject matter relates to compositions comprisingantibodies. It would be understood by one of ordinary skill in the artafter review of the instant disclosure that the presently disclosedsubject matter thus encompasses compositions that consist essentially ofthe antibodies of the presently disclosed subject matter, as well ascompositions that consist of the antibodies of the presently disclosedsubject matter.

As used herein, the phrase “immune cell” refers to the cells of amammalian immune system including but not limited to antigen presentingcells, B cells, basophils, cytotoxic T cells, dendritic cells,eosinophils, granulocytes, helper T cells, leukocytes, lymphocytes,macrophages, mast cells, memory cells, monocytes, natural killer cells,neutrophils, phagocytes, plasma cells and T cells.

As used herein, the phrase “immune response” refers to immunitiesincluding but not limited to innate immunity, humoral immunity, cellularimmunity, immunity, inflammatory response, acquired (adaptive) immunity,autoimmunity, and/or overactive immunity.

As used herein, the phrase “gastrin-associated cancer” is a tumor orcancer or a cell therefrom in which a gastrin gene product acts as atrophic hormone to stimulate tumor and/or cancer cell growth both whenexogenously applied to tumor and/or cancer cells and also in vivothrough autocrine and paracrine mechanisms. Exemplary gastrin-associatedcancers include pancreatic cancer, gastric cancer, gastroesophagealcancer, and colorectal cancer.

The term “polynucleotide” as used herein includes but is not limited toDNA, RNA, complementary DNA (cDNA), messenger RNA (mRNA), ribosomal RNA(rRNA), small hairpin RNA (shRNA), small nuclear RNA (snRNA), shortnucleolar RNA (snoRNA), microRNA (miRNA), genomic DNA, synthetic DNA,synthetic RNA, and/or tRNA.

As used herein, the phrases “single chain variable fragment”,“single-chain antibody variable fragments”, and “scFv” antibodies referto forms of antibodies comprising the variable regions of only the heavyand light chains, connected by a linker peptide.

The term “subject” as used herein refers to a member of any invertebrateor vertebrate species. Accordingly, the term “subject” is intended toencompass in some embodiments any member of the Kingdom Animaliaincluding, but not limited to the phylum Chordata (e.g., members ofClasses Osteichythyes (bony fish), Amphibia (amphibians), Reptilia(reptiles), Ayes (birds), and Mammalia (mammals), and all Orders andFamilies encompassed therein.

The compositions and methods of the presently disclosed subject matterare particularly useful for warm-blooded vertebrates. Thus, in someembodiments the presently disclosed subject matter concerns mammals andbirds. More particularly provided are compositions and methods derivedfrom and/or for use in mammals such as humans and other primates, aswell as those mammals of importance due to being endangered (such asSiberian tigers), of economic importance (animals raised on farms forconsumption by humans) and/or social importance (animals kept as pets orin zoos) to humans, for instance, carnivores other than humans (such ascats and dogs), swine (pigs, hogs, and wild boars), ruminants (such ascattle, oxen, sheep, giraffes, deer, goats, bison, and camels), rodents(such as mice, rats, and rabbits), marsupials, and horses. Also providedis the use of the disclosed methods and compositions on birds, includingthose kinds of birds that are endangered, kept in zoos, as well as fowl,and more particularly domesticated fowl, e.g., poultry, such as turkeys,chickens, ducks, geese, guinea fowl, and the like, as they are also ofeconomic importance to humans. Thus, also provided is the use of thedisclosed methods and compositions on livestock, including but notlimited to domesticated swine (pigs and hogs), ruminants, horses,poultry, and the like.

As used herein, the terms “T cell” and “T lymphocyte” areinterchangeable and used synonymously. Examples include, but are notlimited to, naive T cells, central memory T cells, effector memory Tcells, cytotoxic T cells, T regulatory cells, helper T cells andcombinations thereof.

As used herein, the phrase “therapeutic agent” refers to an agent thatis used to, for example, treat, inhibit, prevent, mitigate the effectsof, reduce the severity of, reduce the likelihood of developing, slowthe progression of, and/or cure, a disease or disorder such as but notlimited to a gastrin-associated tumor and/or cancer.

The terms “treatment” and “treating” as used herein refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) the targeted pathologiccondition, prevent the pathologic condition, pursue or obtain beneficialresults, and/or lower the chances of the individual developing acondition, disease, or disorder, even if the treatment is ultimatelyunsuccessful. Those in need of treatment include those already with thecondition as well as those prone to have or predisposed to having acondition, disease, or disorder, or those in whom the condition is to beprevented.

As used herein, the term “tumor” refers to any neoplastic cell growthand/or proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues the initiation, progression, growth,maintenance, of metastasis of which is directly or indirectly influencedby autocrine and/or paracrine action of gastrin. The terms “cancer” and“tumor” are used interchangeably herein and can refer to both primaryand metastasized solid tumors and carcinomas of any tissue in a subject,including but not limited to pancreatic cancer, gastric cancer,gastroesophageal cancer, and colorectal cancer (referred to hereincollectively as “gastrin-associated” tumors and/or cancers). As usedherein, the terms “cancer and “tumor” are also intended to refer tomulticellular tumors as well as individual neoplastic or pre-neoplasticcells. In some embodiments, a cancer or a tumor comprises a cancer ortumor of an epithelial tissue such as, but not limited to a carcinoma.In some embodiments, a tumor is an adenocarcinoma, which in someembodiments is an adenocarcinoma of the pancreas, liver, stomach,esophagus, colon, or rectum, and/or a metastatic cell derived therefrom.In some embodiments, a tumor and/or a cancer is associated withfibrosis, meaning that as a direct or indirect consequence of thedevelopment of the tumor and/or the cancer, one or more regions offibrosis typically develop in the area of the tumor and/or the cancer.

All genes, gene names, and gene products disclosed herein are intendedto correspond to homologs and/or orthologs from any species for whichthe compositions and methods disclosed herein are applicable. Thus, theterms include, but are not limited to genes and gene products fromhumans and mice. It is understood that when a gene or gene product froma particular species is disclosed, this disclosure is intended to beexemplary only, and is not to be interpreted as a limitation unless thecontext in which it appears clearly indicates. Thus, for example, forthe gastrin gene products presented in GENBANK® biosequence databaseAccession No: NP_000796.1, the human amino acid sequence disclosed isintended to encompass homologous and orthologous gastrin genes and geneproducts from other animals including, but not limited to other mammals,fish, amphibians, reptiles, and birds. Also encompassed are any and allnucleotide sequences that encode the disclosed amino acid sequences,including but not limited to those disclosed in the correspondingGENBANK® entries (i.e., NP_000796.1 and NM_000805.4, respectively).

III. DEVELOPMENT OF AN ANTI-GASTRIN VACCINE

A unique approach to tumor-associated antigen-based vaccines has beenundertaken by exploiting the involvement of gastrin as a key autocrineand paracrine growth factor for PC and other gastrointestinal cancers.This approach involves neutralizing gastrin's trophic effects through anactive humoral immunity against gastrin-17 (G17) with a compound called“Polyclonal Antibody Stimulator” or PAS. PAS comprises a 9-amino acidgastrin epitope derived from the N-terminal sequence of G17 that isidentical in mice and humans and conjugated the same to diphtheriatoxoid (DT) through a linker molecule. This compound has been formulatedin an oil-based adjuvant to create PAS. PAS stimulates the production ofspecific and high-affinity polyclonal anti-G17 antibodies, whereas DTalone had no effect (Watson et al., 1996). Preclinical studies wereperformed in several animal models with gastrointestinal (GI) cancerthat are gastrin responsive, including colon cancer (Singh et al., 1986;Smith & Solomon, 1988; Upp et al., 1989; Smith et al., 1996b), gastriccancer (Smith et al., 1998a; Watson et al., 1989), lung cancer (Rehfeldet al., 1989), and pancreatic cancer (Smith et al., 1990; Smith et al.,1991; Smith et al., 1995; Segal et al., 2014).

In animals, PAS-generated anti-G17 antibodies have been shown to reducethe growth and metastasis of gastrointestinal tumors (Watson et al.,1995; Watson et al., 1996; Watson et al., 1999). Both activeimmunizations with PAS and passive immunization with PAS-generatedanti-G17 antibodies (Watson et al., 1999) have been shown to inhibittumor growth in animal models of GI cancers (Watson et al., 1998; Watsonet al., 1999).

A prospective, randomized, double-blind, placebo controlled groupsequential trial of PAS for the treatment of advanced pancreatic cancerwas conducted in human subjects with advanced pancreatic cancer. Theprimary objective of this study was to compare the effect of monotherapyPAS to placebo on patient survival. Overall, 65% of patients generatedan antibody response to PAS. Subjects in the PAS treated group survivedlonger that the placebo group (average 150 days vs. 84 days,respectively; p=0.016). However, when patients were stratified basedupon whether they generated an immune response to PAS (i.e., PASresponders) or did not generate an immune response (i.e., PASnon-responders), survival was significantly increased (p=0.003) in theresponders.

To date, 469 patients with PDAC have been treated with PAS in clinicaltrials. Approximately 90% of these subjects elicited a protectiveantibody titer. Pooled data from four of the studies (PC1, PC2, PC3, andPC6; Brett et al., 2002; Gilliam et al., 2012) showed that responderpatients had a significant increase in median survival days (191 days)compared with non-responder patients (106 days; p=0.0003). Importantly,none of these patients exhibited any evidence of an autoimmune-typereaction that negatively influenced the normal level and function ofgastrin.

PAS is known to elicit a B cell response with generation of neutralizingantibodies to gastrin. However, clinical studies demonstrated that therewere also long-term survivors, which suggested that additionalmechanisms of anti-tumor immunity could also have been responsible.

IV. PAS+CHECK POINT INHIBITOR COMBINATION THERAPIES

IV.A. Generally

PAS administration generates a humoral antibody response and a cellularimmune response to the onco-fetal protein gastrin, which isinappropriately expressed (i.e., overexpressed) in PDAC. Thisinappropriate gastrin expression in PDAC causes an autocrine andparacrine growth-promoting effect. PAS administration with itssubsequent generation of humoral antibodies to gastrin, will helpeliminate this pathological growth-promoting effect. In addition, aPAS-mediated humoral immune response to gastrin will also help reversethe promotion of angiogenesis, circumvention of apoptosis, increase incell migration, and increase in invasive enzyme expression that areassociated with inappropriate gastrin expression (Watson et al., 2006).

PAS comprises 3 subunits. The first subunit is a gastrin epitope, whichin some embodiments is a peptide that comprises amino-terminal aminoacid residues 1-9 of human G17 with a carboxy-terminal seven (7) aminoacid spacer sequence that terminates in a cysteine residue. An exemplarysequence for this first subunit is EGPWLEEEE (SEQ ID NO: 2).

The second subunit of PAS is a linker that covalently links the firstsubunit to the third subunit. In some embodiments, the linker is aε-maleimido caproic acid N-hydroxysuccinamide ester (eMCS), although anylinker, including non-peptide linkers such as but not limited topolyethylene glycol linkers, could be used for this purpose.

The third subunit of PAS is a diphtheria toxoid, which is used as acarrier protein to enhance a humoral response directed against the firstsubunit (in particular, a humoral response directed against the gastricepitope). It is noted, however, that in some embodiments carrierproteins other than diphtheria toxoid could be employed such as but notlimited to tetanus toxoid or bovine serum albumin.

In some embodiments, the three subunits are formulated for intramuscular(i.m.) injection, and the formulation has excellent physical, chemical,and pharmaceutical properties. PAS also elicits a B cell response withgeneration of neutralizing antibodies to gastrin. This is relevant inPDAC, since gastrin increases cellular proliferation, promotesangiogenesis, facilitates circumvention of apoptosis, increases cellmigration, increases invasive enzyme expression, and is associated withfibrosis on the PDAC microenvironment. In accordance with some aspectsof the presently disclosed subject matter, if the actions of gastrin areblocked, CD8⁺ lymphocytes influx into PDAC, rendering it more likely torespond to immune checkpoint therapy (e.g., a T-cell mediated response).As disclosed herein. PAS also elicits a T cell response and CD8+ cellsthat produce cytokines in response to gastrin stimulation.

PAS can be designed as a therapeutic vaccine or immunotherapeutic.PAS-induced humoral antibodies are highly specific and typicallycharacterized by high affinity to G17 and Gly-G17.

PAS consistently induced therapeutically efficacious levels ofantibodies that are directed against the hormone G17 and its precursorG17-Gly. Twenty-two clinical studies have been completed with a total of1,542 patients. Importantly, treatment with PAS demonstrated anexcellent safety and tolerability profile, and further resulted in asurvival benefit in colorectal, gastric, and pancreatic cancer patients.Used as a monotherapy, an exemplary dose and schedule were identified tobe 250 μg/0.2 m I dosed at 0, 1, and 3 weeks.

Taken collectively, the conclusions that can be made from the 22 studiesand >1,500 patients treated with PAS are as follows:

-   -   (a) Nonclinical data demonstrated both in vitro and in vivo        anti-tumor efficacy of anti-G17 antibodies, with a wide        therapeutic index in various cancer models, including human        pancreatic cancer models;    -   (b) PAS can be administered at very safe and well tolerated        doses, and effectively causes a B cell antibody response to        gastrin with no adverse reactions and no induction of negative        autoimmune effects; and    -   (c) Numerous clinical studies have demonstrated a survival        benefit across gastrointestinal tumors, including pancreatic        cancer, and a correlation between generation of anti-G17        antibody response and improved survival.

However, clinical studies have also demonstrated that there were longterm survivors, which suggested that additional therapeutic benefitsalso resulted from PAS administration. While not wishing to be bound byany particular theory of operation, it is possible that PAS treatmentmight have also induced a T cell immune response characterized byactivation of cytotoxic T cells and memory cells in these subjects.

The use of check point inhibitors in PDAC has been limited, and onlymodest results have been demonstrated. CTLA-4, PD-1, and PD-L1inhibitors have been investigated in patients with locally advanced ormetastatic PDAC in a number of clinical trials (Royal et al., 2010;Brahmer et al., 2012; Segal et al., 2014). Durvalumab (MEDI 4736) hasgenerated a partial response rate of 8% in a preliminary analysis thatwas presented at the American Society of Clinical Oncology in 2014(Segal et al., 2014).

It is not known why pancreatic tumors have proven to be relativelyresistant to monoclonal antibody (mAb)-based immunotherapeutics thattarget check point inhibitors. The failure of anti-immune checkpointinhibitor immunotherapeutics might be related to massive infiltration ofimmunosuppressive leukocytes, which could actually suppress ananti-tumor immune response. This might be related to expression of theRAS oncogene, which drives an inflammatory program that helps establishimmune privilege in the pancreatic tumor microenvironment (Zheng et al.,2013).

IV.B. Check-Point Inhibitors Generate a Cellular Cytotoxic T CellResponse

The immune system has the key central role in differentiating betweenself (i.e., “normal” cells) and “non-self” or “foreign” cells, whetherthis be bacteria found in infections or altered and/or transformed cellsthat are typically found in tumors and cancers. With respect to thisprocess, the immune system requires exquisite regulation to “turn off”when it recognizes “self” so it does not mount an autoimmune reaction tonormal body cells while also needing to “turn on” when it recognizesforeign and/or transformed cells. In fact, cell transformation is arelatively common event, but the immune system keeps efficient andeffective surveillance on this to effectively and efficiently eliminateforeign and/or transformed cells. Tumor formation and cancer arerelatively rare events, since it is only on rare occasions thattransformed cells develop mechanisms where they can subvert normalimmune system checkpoints, resulting in the immune system notrecognizing them as transformed, thus avoiding immune attack by, forexample, cytotoxic T lymphocyte attachment to the transformed tumorcells.

Programmed cell Death protein 1 (PD-1; also known as CD279) is a cellsurface receptor that serves as a checkpoint that is found on thesurface of T cells. PD-1 appears to function as an “off switch” so thatT cells do not mount a cytotoxic T lymphocyte attack against normalcells in the body. Human PD-1 is produced as a 288 amino acid precursorprotein, an exemplary amino acid sequence for which is provided asAccession No. NP_005009.2 of the GENBANK® biosequence database (encodedby GENBANK® Accession No. NM_005018.2). The 288 amino acid precursorincludes a signal peptide as amino acids 1-20 of GENBANK® Accession No.NP_005009.2, which is removed to produce the mature peptide (i.e., aminoacids 21-288 of GENBANK® Accession No. NP_005009.2). The amino acidsequences of orthologs of human PD-1 from other species that are presentin the GENBANK® biosequence database include, but are not limited toAccession Nos. NP_032824.1 (Mus musculus), NP_001100397.1 (Rattusnorvegicus), NP_001301026.1 (Canis lupus familiaris), NP_001138982.1(Felis catus), NP_001076975.1 (Bos taurus), XP_004033550.1 (Gorillagorilla gorilla), NP_001107830.1 (Macaca mulatta), NP_001271065.1(Macaca fascicularis), and XP_003776178.1 (Pongo abelii).

The ligand for the PD-1 receptor is referred to as the Programmeddeath-ligand 1 (PD-L1). It is also known as CD274 or the B7 homolog 1(B7-H1). In humans, there are several isoforms of the PD-L1 protein, thelargest of which (isoform a) is produced as a 290 amino acid precursor.An exemplary amino acid sequence for a human PD-L1 precursor a proteinis provided as Accession No. NP_054862.1 of the GENBANK® biosequencedatabase (encoded by GENBANK® Accession No. NM_014143.3). The 290 aminoacid precursor includes a signal peptide as amino acids 1-18 of GENBANK®Accession No. NP_054862.1, which is removed to produce the maturepeptide (i.e., amino acids 19-290 of GENBANK® Accession No.NP_054862.1). The amino acid sequences of orthologs of human PD-L1 fromother species that are present in the GENBANK® biosequence databaseinclude, but are not limited to Accession Nos. NP_068693.1 (Musmusculus), NP_001178883.1 (Rattus norvegicus), NP_001278901.1 (Canislupus familiaris), XP_006939101.1 (Felis catus), NP_001156884.1 (Bostaurus), XP_018889139.1 (Gorilla gorilla gorilla), NP_001077358.1(Macaca mulatta), XP_015292694.1 (Macaca fascicularis), andXP_009454557.1 (Pongo troglodytes).

PD-L1 is found mainly on normal cells, and when a PD-1 expressing T cellbinds to a normal cell with PD-L1, it signals to the T cell that this isa normal cell (i.e., “self”) and a cytotoxic T cell response against the(normal) cell is suppressed. Most transformed cells are routinelyeliminated since they typically do not express PD-L1, meaning that aPD-1 expressing T cell would not be “shut down” but rather “activated”when encountering such a cell, thereby eliminating that transformedcell. However, on rare occasions, the transformed cell does expressesthe PD-L1 ligand, resulting in a shutdown of a T cell response to thetransformed cell. Hence, transformed cells that express PD-L1 can evadecytotoxic T cell responses. When this occurs, the unrecognizedtransformed cell can expand, acquire additional mutations, and grow intoa malignant, metastatic tumor.

Inhibition of the PD-1/PD-L1 checkpoint (referred to as “immunecheckpoint inhibitors”) can interfere with PD-1/PD-L1 binding, therebyallowing T lymphocytes to recognize tumor and/or cancer cells asnon-self, resulting in cytotoxic T lymphocyte response against the tumorand/or cancer cells. This can be accomplished by drugs that eithertarget PD-1 on T cells or PD-L1 on tumor and/or cancer cells toeffectively block PD-1/PD-L1 interactions. Critical to this process areat least two requirements. First, the immune checkpoint inhibitors mustget to the site of the tumor and/or cancer to block any interactionbetween PD-1 and PD-L1. Second, the tumor and/or cancer itself must beaccessible to cytotoxic T cells.

Another checkpoint protein is the cytotoxic T-lymphocyte antigen 4(CTLA-4; also known as CD152) protein. Like PD-1, CTLA-4 is a cellsurface receptor that can downregulate immune responses. T_(regs)express CTLA-4, as do activated T cells. When the CTLA-4 receptor bindsto CD80 or CD86 present on the surface of antigen-presenting cells(APCs), like PD-1 it functions as an “off switch” with respect to immuneresponses.

The human CTLA4-TM isoform is a 223 amino acid precursor protein thathas the amino acid sequence set forth in GENBANK® Accession No.NP_005205.2 (encoded by GENBANK® Accession No. NM_005214.4). Thisprotein includes a 35 amino acid signal peptide, that when removedgenerates the 188 amino acid mature peptide. The amino acid sequences oforthologs of human CTLA-4 from other species that are present in theGENBANK® biosequence database include, but are not limited to AccessionNos. NP_033973.2 (Mus musculus), NP_113862.1 (Rattus norvegicus),NP_001003106.1 (Canis lupus familiaris), NP_001009236.1 (Felis catus),NP_776722.1 (Bos taurus), XP_004033133.1 (Gorilla gorilla gorilla),XP_009181095.2 (Macaca mulatta), XP_005574073.1 (Macaca fascicularis),and XP_526000.1 (Pan troglodytes).

As such, in some embodiments the presently disclosed subject matterpertains to the administration of PAS with one or more immune checkpoint inhibitors. More particularly, in some embodiments the presentlydisclosed subject matter relates to use of immune checkpoint inhibitorsthat target CTLA-4, PD-1, and/or PD-L1. Exemplary compounds that inhibitthese immune checkpoint inhibitors include the following. For CTLA-4:Ipilimumab (YERVOY® brand; Bristol-Myers Squibb, New York, N.Y.) andTremelimumab (formerly Ticilimumab; Medimmune, LLC, Gaithersburg, Md.For PD-1: Nivolumab (OPDIVO® brand; Bristol-Myers Squibb, New York,N.Y.), Pidilizumab (Medivation, San Francisco, Calif.), Pembrolizumab(KEYTRUDA® brand; Merck & Co., Inc., Kenilworth, N.J.), MEDI0680(AMP514; Medimmune, LLC, Gaithersburg, Md.), and AUNP-12 (AurigeneDiscovery Technologies Limited/Laboratoires Pierre Fabre SA). For PD-L1:BMS-936559/MDX-1105 (Bristol Myers Squibb, New York, N.Y.), Atezolizumab(TECENTRIQ® brand; Genentech/Roche, South San Francisco, Calif.),Durvalumab (MEDI4736; Medimmune, LLC, Gaithersburg, Md.), and Avelumab(BAVENCIO® brand; EMD Serono, Inc., Rockland, Md., and Pfizer Inc., NewYork, N.Y.).

Evidence strongly suggests there is more commonality rather thandivergence related to efficacy and toxicity when one compares the PD-1and PD-L1 inhibitors. In fact, in cross-trial meta-type analyses,Nivolumab, Pembrolizumab, Avelumab, Atezolizumab, and MDX1105 have beenshown to have very similar (but not identical) profiles with respect totoxicity and efficacy. Although affinities and current dosing regimensmight differ for the various PD-1 and PD-L1 inhibitors, there isgenerally a very wide therapeutic window for all. Associated with thisbroad therapeutic window is the observation that most of these checkpoint inhibitors do not fail in Phase I and many clinical developmentplans are moving to flat dosing regiments rather than metered dosing.

Although the drugs that target PD-1 and PD-L1 have similar modes ofaction, efficacy profiles, and toxicity profiles, in general there aresome subtle differences between them. Avelumab might have some advantageover other PD-L1 targeted drugs due to capability to complement patentapplications-derived B cell responses with an antibody-dependentcell-mediated cytotoxicity (ADDC) response. Avelumab also has a nativeFc receptor and therefore can elicit a “normal” ADCC response, whereasAtezolizumab has modifications in the Fc region that might be expectedto reduce the ADCC response (at least in humans).

Another difference to note in comparing different PD-1 and PD-L1targeted drugs is the fact that some are humanized mAbs while others arefully human mAbs. Humanized mAbs might be expected to be characterizedby an increased likelihood of inducing “allergic” type reactionscompared to fully human mAbs when the humanized mAbs are administered tohumans.

V. COMPOSITIONS

V.A. Pharmaceutical Compositions

In some embodiments, the presently disclosed subject matter providespharmaceutical compositions that in some embodiments can be employed inthe methods of the presently disclosed subject matter.

As used herein, a “pharmaceutical composition” refers to a compositionthat is to be employed as part of a treatment or other method whereinthe pharmaceutical composition will be administered to a subject in needthereof. In some embodiments, a subject in need thereof is a subjectwith a tumor and/or a cancer at least one symptom, characteristic, orconsequence of which is expected to be ameliorated at least in part dueto a biological activity of the pharmaceutical composition actingdirectly and/or indirectly on the tumor and/or the cancer and/or a cellassociated therewith.

Techniques for preparing pharmaceutical compositions are known in theart, and in some embodiments pharmaceutical compositions are formulatedbased on the subject to which the pharmaceutical compositions are to beadministered. For example, in some embodiments a pharmaceuticalcomposition is formulated for use in a human subject. Thus, in someembodiments a pharmaceutical composition is pharmaceutically acceptablefor use in a human.

The pharmaceutical compositions of the presently disclosed subjectmatter in some embodiments comprise a first agent that induces and/orprovides an active and/or a passive humoral immune response against agastrin peptide and/or a CCK-B receptor; and an immune checkpointinhibitor. In some embodiments, the first agent is selected from thegroup consisting of a gastrin peptide, an anti-gastrin antibody, and ananti-CCK-R antibody. In some embodiments, the first agent comprises agastrin peptide, optionally a gastrin peptide comprising, consistingessentially of, or consisting of an amino acid sequence selected fromthe group consisting of EGPWLEEEEE (SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO:2), EGPWLEEEEEAY (SEQ ID NO: 3), and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4).In some embodiments, the glutamic acid residue an amino acid position 1of any of SEQ ID NOs: 1-4 is a pyroglutamate residue. In someembodiments, the gastrin peptide is conjugated to an immunogeniccarrier, optionally wherein the immunogenic carrier is selected from thegroup consisting of diphtheria toxoid, tetanus toxoid, keyhole limpethemocyanin, and bovine serum albumin. In some embodiments, the gastrinpeptide is conjugated to an immunogenic carrier via a linker, optionallywherein the linker comprises a ε-maleimido caproic acidN-hydroxysuccinamide ester.

In some embodiments, the linker and the gastrin peptide are separated byan amino acid spacer, optionally wherein the amino acid spacer isbetween 1 and 10 amino acids in length, further optionally wherein theamino acid spacer is 7 amino acids in length.

Pharmaceutical compositions of the presently disclosed subject matterthat are designed to elicit humoral immune responses can in someembodiments further comprise an adjuvant, optionally an oil-basedadjuvant. Exemplary adjuvants include but are not limited to montanideISA-51 (Seppic, Inc.); QS-21 (Aquila Pharmaceuticals, Inc.); Arlacel A;oeleic acid; tetanus helper peptides; GM-CSF; cyclophosamide; bacillusCalmette-Guerin (BCG); corynbacterium parvum; levamisole, azimezone;isoprinisone; dinitrochlorobenezene (DNCB); keyhole limpet hemocyanins(KLH) including Freunds adjuvant (complete and incomplete); mineralgels; aluminum hydroxide (Alum); lysolecithin; pluronic polyols;polyanions; peptides; oil emulsions; nucleic acids (e.g., dsRNA)dinitrophenol; diphtheria toxin (DT); toll-like receptor (TLR, e.g.,TLR3, TLR4, TLR7, TLR8 or TLR9) agonists (e.g, endotoxins such aslipopolysaccharide (LPS); monophosphoryl lipid A (MPL);polyinosinic-polycytidylic acid (poly-ICLC/HILTONOL®; Oncovir, Inc.,Washington, D.C., United States of America); IMO-2055, glucopyranosyllipid A (GLA), QS-21—a saponin extracted from the bark of the Quillajasaponaria tree, also known as the soap bark tree or Soapbark; resiquimod(TLR7/8 agonist), CDX-1401—a fusion protein consisting of a fully humanmonoclonal antibody with specificity for the dendritic cell receptorDEC-205 linked to the NY-ESO-1 tumor antigen; Juvaris' CationicLipid-DNA Complex; Vaxfectin; and combinations thereof.

The pharmaceutical compositions of the presently disclosed subjectmatter can in some embodiments comprise an immune checkpoint inhibitor.Immune checkpoint inhibitors are a class of compounds that inhibits abiological activity of a target polypeptide selected from the groupconsisting of cytotoxic T-lymphocyte antigen 4 (CTLA4), programmed celldeath-1 receptor (PD-1), and programmed cell death 1 receptor ligand(PD-L1). In some embodiments, the immune checkpoint inhibitor isselected from the group consisting of Ipilimumab, Tremelimumab,Nivolumab, Pidilizumab, Pembrolizumab, AMP514, AUNP12,BMS-936559/MDX-1105, Atezolizumab, MPDL3280A, RG7446, R05541267,MEDI4736, Avelumab and Durvalumab.

In some embodiments of the presently disclosed pharmaceuticalcompositions, the first agent comprises an amount of a gastrin peptidecomprising, consisting essentially of, or consisting of an amino acidsequence selected from the group consisting of EGPWLEEEEE (SEQ ID NO:1), EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY (SEQ ID NO: 3), andEGPWLEEEEEAYGWMDF (SEQ ID NO: 4) effective to induce an anti-gastrinhumoral response and the second agent comprises an amount of acheckpoint inhibitor that is effective to induce or enhance a cellularimmune response against a gastrin-associated tumor or cancer whenadministered to a subject who has gastrin-associated tumor or cancer.

In some embodiments of the presently disclosed pharmaceuticalcompositions, the first agent comprises one or more anti-CCK-B receptorantibodies and is present in the pharmaceutical composition in an amountsufficient to reduce or inhibit gastrin signaling via CCK-B receptorspresent on a gastrin-associated tumor or cancer when administered to asubject that has a gastrin-associated tumor or cancer.

The pharmaceutical compositions of the presently disclosed subjectmatter are in some embodiments employed to treat a gastrin-associatedtumor and/or cancer. In some embodiments, pharmaceutical compositions ofthe presently disclosed subject matter are intended to treat pancreaticcancer.

V.B. Nucleic Acids

The term “RNA” refers to a molecule comprising at least oneribonucleotide residue. By “ribonucleotide” is meant a nucleotide with ahydroxyl group at the 2′ position of a β-D-ribofuranose moiety. Theterms encompass double stranded RNA, single stranded RNA, RNAs with bothdouble stranded and single stranded regions, isolated RNA such aspartially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA, or analog RNA, thatdiffers from naturally occurring RNA by the addition, deletion,substitution, and/or alteration of one or more nucleotides. Suchalterations can include addition of non-nucleotide material, such as tothe end(s) of an siRNA or internally, for example at one or morenucleotides of the RNA. Nucleotides in the RNA molecules of thepresently disclosed subject matter can also comprise non-standardnucleotides, such as non-naturally occurring nucleotides or chemicallysynthesized nucleotides or deoxynucleotides. These altered RNAs can bereferred to as analogs or analogs of a naturally occurring RNA.

The terms “small interfering RNA”, “short interfering RNA”, “smallhairpin RNA”, “siRNA”, and shRNA are used interchangeably and refer toany nucleic acid molecule capable of mediating RNA interference (RNAi)or gene silencing. See e.g., Bass, Nature 411:428-429, 2001; Elbashir etal., Nature 411:494-498, 2001a; and PCT International Publication Nos.WO 00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO 01/29058, WO99/07409, and WO 00/44914. In one embodiment, the siRNA comprises adouble stranded polynucleotide molecule comprising complementary senseand antisense regions, wherein the antisense region comprises a sequencecomplementary to a region of a target nucleic acid molecule (forexample, a nucleic acid molecule encoding a gastrin gene product). Inanother embodiment, the siRNA comprises a single stranded polynucleotidehaving self-complementary sense and antisense regions, wherein theantisense region comprises a sequence complementary to a region of atarget nucleic acid molecule. In another embodiment, the siRNA comprisesa single stranded polynucleotide having one or more loop structures anda stem comprising self-complementary sense and antisense regions,wherein the antisense region comprises a sequence complementary to aregion of a target nucleic acid molecule, and wherein the polynucleotidecan be processed either in vivo or in vitro to generate an active siRNAcapable of mediating RNAi. As used herein, siRNA molecules need not belimited to those molecules containing only RNA, but further encompasschemically modified nucleotides and non-nucleotides.

The presently disclosed subject matter takes advantage of the ability ofshort, double stranded RNA molecules to cause the down regulation ofcellular genes, a process referred to as RNA interference. As usedherein, “RNA interference” refers to a process of sequence-specificpost-transcriptional gene silencing mediated by a small interfering RNA(siRNA). See generally Fire et al., Nature 391:806-811, 1998. Theprocess of post-transcriptional gene silencing is thought to be anevolutionarily conserved cellular defense mechanism that has evolved toprevent the expression of foreign genes (Fire, Trends Genet 15:358-363,1999).

RNAi might have evolved to protect cells and organisms against theproduction of double stranded RNA (dsRNA) molecules resulting frominfection by certain viruses (particularly the double stranded RNAviruses or those viruses for which the life cycle includes a doublestranded RNA intermediate) or the random integration of transposonelements into the host genome via a mechanism that specifically degradessingle stranded RNA or viral genomic RNA homologous to the doublestranded RNA species.

The presence of long dsRNAs in cells stimulates the activity of theenzyme Dicer, a ribonuclease III. Dicer catalyzes the degradation ofdsRNA into short stretches of dsRNA referred to as small interferingRNAs (siRNA; Bernstein et al., Nature 409:363-366, 2001). The smallinterfering RNAs that result from Dicer-mediated degradation aretypically about 21-23 nucleotides in length and contain about 19 basepair duplexes. After degradation, the siRNA is incorporated into anendonuclease complex referred to as an RNA-induced silencing complex(RISC). The RISC is capable of mediating cleavage of single stranded RNApresent within the cell that is complementary to the antisense strand ofthe siRNA duplex. According to Elbashir et al., cleavage of the targetRNA occurs near the middle of the region of the single stranded RNA thatis complementary to the antisense strand of the siRNA duplex (Elbashiret al., Genes Dev 15:188-200, 2001b).

RNAi has been described in several cell type and organisms. Fire et al.,1998 described RNAi in C. elegans. Wianny & Zernicka-Goetz, Nature CellBiol 2:70-75, 1999 disclose RNAi mediated by dsRNA in mouse embryos.Hammond et al., Nature 404:293-296, 2000 were able to induce RNAi inDrosophila cells by transfecting dsRNA into these cells. Elbashir et al.Nature 411:494-498, 2001a demonstrated the presence of RNAi in culturedmammalian cells including human embryonic kidney and HeLa cells by theintroduction of duplexes of synthetic 21 nucleotide RNAs.

Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of a siRNA duplex facilitate siRNA activityand that ATP is utilized to maintain the 5′-phosphate moiety on thesiRNA (Nykanen et al., Cell 107:309-321, 2001). Other modifications thatmight be tolerated when introduced into an siRNA molecule includemodifications of the sugar-phosphate backbone or the substitution of thenucleoside with at least one of a nitrogen or sulfur heteroatom (PCTInternational Publication Nos. WO 00/44914 and WO 01/68836) and certainnucleotide modifications that might inhibit the activation of doublestranded RNA-dependent protein kinase (PKR), specifically 2′-amino or2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge (Canadian Patent Application No. 2,359,180).

Other references disclosing the use of dsRNA and RNAi include PCTInternational Publication Nos. WO 01/75164 (in vitro RNAi system usingcells from Drosophila and the use of specific siRNA molecules forcertain functional genomic and certain therapeutic applications); WO01/36646 (methods for inhibiting the expression of particular genes inmammalian cells using dsRNA molecules); WO 99/32619 (methods forintroducing dsRNA molecules into cells for use in inhibiting geneexpression); WO 01/92513 (methods for mediating gene suppression byusing factors that enhance RNAi); WO 02/44321 (synthetic siRNAconstructs); WO 00/63364 and WO 01/04313 (methods and compositions forinhibiting the function of polynucleotide sequences); and WO 02/055692and WO 02/055693 (methods for inhibiting gene expression using RNAi).

In some embodiments, the presently disclosed subject matter utilizesRNAi to at least partially inhibit expression of at least one gastringene product. Inhibition is preferably at least about 10% of normalexpression amounts. In some embodiments, the method comprisesintroducing an RNA to a target cell in an amount sufficient to inhibitexpression of a gastrin gene product, wherein the RNA comprises aribonucleotide sequence which corresponds to a coding strand of a geneof interest. In some embodiments, the target cell is present in asubject, and the RNA is introduced into the subject.

The RNA can have a double-stranded region comprising a first strandcomprising a ribonucleotide sequence that corresponds to the codingstrand of the gene encoding the target protein (for example, a gastringene product) and a second strand comprising a ribonucleotide sequencethat is complementary to the first strand. The first strand and thesecond strand hybridize to each other to form the double-strandedmolecule. The double stranded region can be at least 15 basepairs inlength, and in some embodiments, between 15 and 50 basepairs in length,and in some embodiments the double stranded region is between 15 and 30basepairs in length.

In some embodiments, the RNA comprises one strand that forms adouble-stranded region by intramolecular self-hybridization, which ispreferably complementary over at least 19 bases. In some embodiments,the RNA comprises two separate strands that form a double-strandedregion by intermolecular hybridization that is complementary over atleast 19 bases.

One skilled in the art will recognize that any number of suitable commontechniques can be used to introduce the RNAs into a target cell. In someembodiments, a vector encoding the RNA is introduced to the target cell.For example, the vector encoding the RNA can be transfected into thetarget cell and the RNA is then transcribed by cellular polymerases.

In some embodiments, a recombinant virus comprising nucleic acidencoding the RNA can be produced. Introducing the RNA into a target cellthen comprises infecting the target cell with the recombinant virus.Cellular polymerases transcribe the RNA resulting in expression of theRNA within the target cell. Engineering recombinant viruses is wellknown to those having ordinary skill in the art. One of skill wouldreadily appreciate the multiple factors involved in selecting theappropriate virus and vector components needed to optimize recombinantvirus production for use with the presently disclosed subject matterwithout the necessity of further detailed discussion herein. As onenon-limiting example, a recombinant adenovirus can be engineeredcomprising DNA encoding an siRNA. The virus can be engineered to bereplication deficient such that cells can be infected by the recombinantadenovirus, the siRNA transcribed, and transiently expressed in theinfected target cell. Details of recombinant virus production and usecan be found in PCT International Patent Application Publication No. WO2003/006477, herein incorporated by reference in their entireties.Alternatively, a commercial kit for producing recombinant viruses can beused, such as for example, the pSILENCER ADENO 1.0-CMV SYSTEM™ brandvirus production kit (Ambion, Austin, Tex., United States of America).

V.C. Gene Editing

Downregulation of gene products can also be accomplished using theCRISPR-Cas gene editing system as described in U.S. Pat. No. 8,945,839to Zhang and references cited therein, Al-Attar et al., 2011; Makarovaet al., 2011; Le Cong et al., 2013; Seung Woo Cho et al., 2013a, b;Carroll, 2012; Gasiunas et al., 2012; Hale et al., 2012; and Jinek etal., 2012, all of which are incorporated herein by reference in theirentireties. In some embodiments, the methods and compositions for use inthe CRISPR-Cas gene editing system include nucleic acids that target agastrin gene sequence, which in some embodiments is a gastrin genesequence in a tumor and/or a cancer.

V.D. Formulations

Compositions as described herein comprise in some embodiments acomposition that includes a pharmaceutically acceptable carrier.Suitable formulations include aqueous and non-aqueous sterile injectionsolutions that can contain antioxidants, buffers, bacteriostats,bactericidal antibiotics, and solutes that render the formulationisotonic with the bodily fluids of the intended recipient; and aqueousand non-aqueous sterile suspensions, which can include suspending agentsand thickening agents. In some embodiments, a formulation of thepresently disclosed subject matter comprises an adjuvant, optionally anoil-based adjuvant.

The compositions used in the methods can take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing, and/or dispersingagents. The compositions used in the methods can take forms including,but not limited to perioral, intravenous, intraperitoneal,intramuscular, and intratumoral formulations. Alternatively or inaddition, the active ingredient can be in powder form for constitutionwith a suitable vehicle (e.g., sterile pyrogen-free water) before use.

The formulations can be presented in unit-dose or multi-dose containers,for example sealed ampules and vials, and can be stored in a frozen orfreeze-dried (lyophilized) condition requiring only the addition ofsterile liquid carrier immediately prior to use.

For oral administration, the compositions can take the form of, forexample, tablets or capsules prepared by a conventional technique withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets can be coated by methods known in the art. For example, aneuroactive steroid can be formulated in combination withhydrochlorothiazide, and as a pH stabilized core having an enteric ordelayed-release coating which protects the neuroactive steroid until itreaches the colon.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional techniqueswith pharmaceutically acceptable additives such as suspending agents(e.g., sorbitol syrup, cellulose derivatives or hydrogenated ediblefats); emulsifying agents (e.g. lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring, and sweetening agents asappropriate. Preparations for oral administration can be suitablyformulated to give controlled release of the active compound. For buccaladministration the compositions can take the form of tablets or lozengesformulated in conventional manner.

The compounds can also be formulated as a preparation for implantationor injection. Thus, for example, the compounds can be formulated withsuitable polymeric or hydrophobic materials (e.g., as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives (e.g., as a sparingly soluble salt).

The compounds can also be formulated in oils that are administered aswater-in-oil emulsions, oil-in-water emulsions, or water-in-oil-in wateremulsions.

The compounds can also be formulated in rectal compositions (e.g.,suppositories or retention enemas containing conventional suppositorybases such as cocoa butter or other glycerides), creams or lotions, ortransdermal patches.

In some embodiments, the presently disclosed subject matter employs acomposition that is pharmaceutically acceptable for use in humans. Oneof ordinary skill in the art understands the nature of those componentsthat can be present in such a composition that is pharmaceuticallyacceptable for use in humans and also what components should be excludedfrom compositions that are pharmaceutically acceptable for use inhumans.

V.E. Doses

As used herein, the phrases “treatment effective amount”,“therapeutically effective amount”, “treatment amount”, and “effectiveamount” are used interchangeably and refer to an amount of a therapeuticcomposition sufficient to produce a measurable response (e.g., abiologically or clinically relevant response in a subject beingtreated). Actual dosage levels of active ingredients in thepharmaceutical compositions of the presently disclosed subject mattercan be varied so as to administer an amount of the active compound(s)that is effective to achieve the desired therapeutic response for aparticular subject. The selected dosage level can depend upon theactivity of the therapeutic composition, the route of administration,combination with other drugs or treatments, the severity of thecondition being treated, the condition and prior medical history of thesubject being treated, etc. However, it is within the skill of the artto start doses of the compound at levels lower than required to achievethe desired therapeutic effect and to gradually increase the dosageuntil the desired effect is achieved.

The potency of a therapeutic composition can vary, and therefore a“therapeutically effective amount” can vary. However, one skilled in theart can readily assess the potency and efficacy of a candidate modulatorof the presently disclosed subject matter and adjust the therapeuticregimen accordingly.

After review of the disclosure herein of the presently disclosed subjectmatter, one of ordinary skill in the art can tailor the dosages to anindividual subject, taking into account the particular formulation,method of administration to be used with the composition, and otherfactors. Further calculations of dose can consider subject height andweight, severity and stage of symptoms, and the presence of additionaldeleterious physical conditions. Such adjustments or variations, as wellas evaluation of when and how to make such adjustments or variations,are well known to those of ordinary skill in the art of medicine.

Thus, in some embodiments the term “effective amount” is used herein torefer to an amount of a composition comprising an agent that providesand/or induces a humoral or cellular immune response against a gastrinpeptide and or comprising a nucleic acid that inhibits expression of agastrin gene product, a pharmaceutically acceptable salt thereof, aderivative thereof, or a combination thereof sufficient to produce ameasurable anti-tumor and/or anti-cancer biological activity. Actualdosage levels of active ingredients in composition of the presentlydisclosed subject matter can be varied so as to administer an amount ofthe active compound(s) that is effective to achieve the desired responsefor a particular subject and/or application. The selected dosage levelcan depend upon a variety of factors including the activity of thecomposition, formulation, route of administration, combination withother drugs or treatments, severity of the condition being treated, andphysical condition and prior medical history of the subject beingtreated. In some embodiments, a minimal dose is administered, and doseis escalated in the absence of dose-limiting toxicity to a minimallyeffective amount. Determination and adjustment of an effective dose, aswell as evaluation of when and how to make such adjustments, are knownto those of ordinary skill in the art.

For administration of a composition as disclosed herein, conventionalmethods of extrapolating human dosage based on doses administered to amurine animal model can be carried out using techniques known to one ofordinary skill in the art. Drug doses can also be given in milligramsper square meter of body surface area because this method rather thanbody weight achieves a good correlation to certain metabolic andexcretionary functions. Moreover, body surface area can be used as acommon denominator for drug dosage in adults and children as well as indifferent animal species as described by Freireich et al., 1966.Briefly, to express a mg/kg dose in any given species as the equivalentmg/m² dose, multiply the dose by the appropriate km factor. In an adulthuman, 100 mg/kg is equivalent to 100 mg/kg×37 kg/m²=3700 mg/m².

For additional guidance regarding formulations and doses, see U.S. Pat.Nos. 5,326,902; 5,234,933; PCT International Publication No. WO93/25521; Remington et al., 1975; Goodman et al., 1996; Berkow et al.,1997; Speight et al., 1997; Ebadi, 1998; Duch et al., 1998; Katzung,2001; Gerbino, 2005.

V.F. Routes of Administration

The presently disclosed compositions can be administered to a subject inany form and/or by any route of administration. In some embodiments, theformulation is a sustained release formulation, a controlled releaseformulation, or a formulation designed for both sustained and controlledrelease. As used herein, the term “sustained release” refers to releaseof an active agent such that an approximately constant amount of anactive agent becomes available to the subject over time. The phrase“controlled release” is broader, referring to release of an active agentover time that might or might not be at a constant level. Particularly,“controlled release” encompasses situations and formulations where theactive ingredient is not necessarily released at a constant rate, butcan include increasing release over time, decreasing release over time,and/or constant release with one or more periods of increased release,decreased release, or combinations thereof. Thus, while “sustainedrelease” is a form of “controlled release”, the latter also includesdelivery modalities that employ changes in the amount of an active agentthat are delivered at different times.

In some embodiments, the sustained release formulation, the controlledrelease formulation, or the combination thereof is selected from thegroup consisting of an oral formulation, a peroral formulation, a buccalformulation, an enteral formulation, a pulmonary formulation, a rectalformulation, a vaginal formulation, a nasal formulation, a lingualformulation, a sublingual formulation, an intravenous formulation, anintraarterial formulation, an intracardial formulation, an intramuscularformulation, an intraperitoneal formulation, a transdermal formulation,an intracranial formulation, an intracutaneous formulation, asubcutaneous formulation, an aerosolized formulation, an ocularformulation, an implantable formulation, a depot injection formulation,a transdermal formulation and combinations thereof. In some embodiments,the route of administration is selected from the group consisting oforal, peroral, buccal, enteral, pulmonary, rectal, vaginal, nasal,lingual, sublingual, intravenous, intraarterial, intracardial,intramuscular, intraperitoneal, transdermal, intracranial,intracutaneous, subcutaneous, ocular, via an implant, and via a depotinjection. Where applicable, continuous infusion can enhance drugaccumulation at a target site (see, e.g., U.S. Pat. No. 6,180,082). Seealso U.S. Pat. Nos. 3,598,122; 5,016,652; 5,935,975; 6,106,856;6,162,459; 6,495,605; and 6,582,724; and U.S. Patent ApplicationPublication No. 2006/0188558 for transdermal formulations and methods ofdelivery of compositions. In some embodiments, the administering is viaa route selected from the group consisting of peroral, intravenous,intraperitoneal, inhalation, and intratumoral.

The particular mode of administration of the compositions of thepresently disclosed subject matter used in accordance with the methodsdisclosed herein can depend on various factors, including but notlimited to the formulation employed, the severity of the condition to betreated, whether the active agents in the compositions (e.g., PAS) areintended to act locally or systemically, and mechanisms for metabolismor removal of the active agents following administration.

VI. METHODS AND USES

In some embodiments, the presently disclosed subject matter relates toemploying pharmaceutical compositions in the context of various methodsand/or uses related to treating gastrin-associated tumors and/orcancers, producing medicaments for treating gastrin-associated tumorsand/or cancers, inhibiting growth of gastrin-associated tumors and/orcancers, inducing and/or enhancing humoral and/or cellular immuneresponses against gastrin-associated tumors and/or cancers, sensitizingtumors and/or cancers associated with gastrin and/or CCK-B receptorsignaling in subjects to inducers of cellular immune responses directedagainst the tumors and/or cancers, preventing, reducing, and/oreliminating formation of fibrosis associated with tumors and/or cancers,particularly in the context of pancreatic cancer; preventing, reducing,and/or eliminating metastases of gastrin-associated tumors and/orcancers; increasing the number of tumor-infiltrating CD8⁺ lymphocytes intumors and/or cancers; reducing the number of FoxP3⁺ inhibitoryT-regulatory cells present in tumors and/or cancers; and increasing thenumber of T_(EMRA) cells in subject that respond to gastrin-associatedtumors and/or cancers. Each of these methods and/or uses is described inmore detail herein below.

VI.A. Methods for Treating Gastrin-associated Tumors and/or Cancers

In some embodiments, the presently disclosed subject matter relates tomethods for treating gastrin-associated tumors and/or cancers. In someembodiments, the method comprises administering to a subject in needthereof (e.g., a subject with a gastrin-associated tumor and/or cancer)an effective amount of a composition that comprises a first agent thatinduces and/or provides an active and/or a passive humoral immuneresponse against a gastrin peptide and/or a CCK-B receptor; and a secondagent that induces and/or provides a cellular immune response againstthe gastrin-associated tumor or cancer. Thus, the presently disclosedmethods in some embodiments rely on the use of pharmaceuticalcompositions that have one or more active agents that together providetwo distinct immunotherapeutic activities: providing and/or inducing anactive and/or a passive humoral immune response against a gastrinpeptide and/or a CCK-B receptor, and inducing and/or providing acellular immune response against the gastrin-associated tumor and/orcancer.

With respect to providing and/or inducing an active and/or a passivehumoral immune response against a gastrin peptide and/or a CCK-Breceptor, the first agent present in the pharmaceutical compositions ofthe presently disclosed subject matter is selected from the groupconsisting of a gastrin peptide designed to induce an active humoralresponse against gastrin, and/or an anti-gastrin antibody and/or ananti-CCK-R antibody designed to provide a passive humoral responseagainst gastrin and/or a CCK-B receptor, in some embodiments a CCK-Breceptor present on gastrin-associated tumor and/or cancer. While notwishing to be bound by any particular theory of action, the activeand/or a passive humoral immune response against a gastrin peptideand/or a CCK-B receptor is designed to inhibit, either partially orcompletely, gastrin signaling in the gastrin-associated tumor and/orcancer via the CCK-B receptor by reducing gastrin binding to the CCK-Breceptor by reducing the amount of circulating gastrin present in thesubject and/or by interfering with gastrin binding to the CCK-B receptorwith neutralizing and/or blocking antibodies.

Thus, in some embodiments the first agent comprises a gastrin peptide,optionally a gastrin peptide comprising, consisting essentially of, orconsisting of an amino acid sequence selected from the group consistingof EGPWLEEEEE (SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY(SEQ ID NO: 3), and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4), wherein theglutamic acid residue at amino acid position 1 of any of SEQ ID NOs: 1-4is a pyroglutamate residue. In some embodiments, the gastrin peptide isconjugated to an immunogenic carrier, optionally via a linker, furtheroptionally a linker comprising a ε-maleimido caproic acidN-hydroxysuccinamide ester, in the pharmaceutical composition.Non-limiting examples of immunogenic carriers include diphtheria toxoid,tetanus toxoid, keyhole limpet hemocyanin, and bovine serum albumin. Thestructure of the first agent is described in more detail herein above,but in some embodiments the linker and the gastrin peptide are separatedby an amino acid spacer, optionally wherein the amino acid spacer isbetween 1 and 10 amino acids in length, further optionally wherein theamino acid spacer is 7 amino acids in length.

As would be appreciated by one of ordinary skill in the art uponconsideration of this disclosure, in some embodiments the pharmaceuticalcomposition further comprises an adjuvant, optionally an oil-basedadjuvant, to enhance the immunogenicity of the gastrin peptide and/orthe gastrin peptide conjugate when an active anti-gastrin humoral immuneresponse is desired.

In order to induce a cellular immune response against thegastrin-associated tumor or cancer, the methods of the presentlydisclosed subject matter employ pharmaceutical compositions thatcomprise one or more checkpoint inhibitors. As is known, checkpointinhibitors inhibit one or more biological activities of targetpolypeptides that have immune checkpoint activities. Exemplary suchpolypeptides include cytotoxic T-lymphocyte antigen 4 (CTLA4)polypeptides, programmed cell death-1 receptor (PD-1) polypeptides, andprogrammed cell death 1 receptor ligand (PD-L1) polypeptides. In someembodiments, a checkpoint inhibitor comprises an antibody or a smallmolecule that binds to and/or interferes with interactions between Tcells and tumor cells by inhibiting or preventing interactions betweenPD-1 polypeptides and PD-L1 polypeptides. Exemplary such antibodies andsmall molecules include but are not limited to Ipilimumab, Tremelimumab,Nivolumab, Pidilizumab, Pembrolizumab, AMP514, AUNP12,BMS-936559/MDX-1105, Atezolizumab, MPDL3280A, RG7446, R05541267,MEDI4736, Avelumab and Durvalumab.

The pharmaceutical compositions of the presently disclosed subjectmatter can include various amounts of the first and second agents,provided that both humoral and cellular responses are induced and/orprovided in the subject, and the amounts of the first and second agentspresent in the pharmaceutical compositions can be adjusted in order tomaximize the effectiveness of the treatment and/or minimize undesirableside effects thereof. However, in some embodiments a pharmaceuticalcomposition of the presently disclosed subject matter is administered ina dose selected from the group consisting of about 50 μg to about 1000μg, about 50 μg to about 500 μg, about 100 μg to about 1000 μg, about200 μg to about 1000 μg, and about 250 μg to about 500 μg, andoptionally wherein the dose is repeated once, twice, or three times,optionally wherein the second dose is administered 1 week after thefirst dose and the third dose, if administered, is administered 1 or 2weeks after the second dose.

In some embodiments, a method for treating a gastrin-associated tumorand/or cancer of the presently disclosed subject matter comprisesadministering to a subject in need thereof a first agent that directlyor indirectly inhibits one or more biological activities of gastrin inthe tumor and/or cancer and a second agent comprising a stimulator of acellular immune response against the tumor and/or the cancer. As such,in some embodiments the first agent directly or indirectly inhibits oneor more biological activities of gastrin in the tumor and/or cancer byproviding and/or inducing a humoral immune response against a gastrinpeptide, optionally wherein the agent is selected from the groupconsisting of an anti-gastrin antibody and a gastrin peptide thatinduces production of neutralizing anti-gastrin antibodies in thesubject; and/or comprises a nucleic acid that inhibits expression of agastrin gene product. Nucleic acids that inhibit expression of a gastringene product would be understood by one of ordinary skill in the artafter consideration of this disclosure, and examples are discussedherein above.

Anti-gastrin antibodies are known in the art and are described in U.S.Pat. Nos. 5,607,676; 5,609,870; 5,622,702; 5,785,970; 5,866,128; and6,861,510. See also PCT International Patent Application PublicationNos. WO 2003/005955 and WO 2005/095459. The content of each of theseU.S. Patents and PCT International Patent Application Publications isincorporated herein in its entirety. In some embodiments, ananti-gastrin antibody is an antibody directed against an epitope presentwithin gastrin-17 (G17). In some embodiments, the epitope is presentwithin one or more of the amino acid sequences EGPWLEEEEE (SEQ ID NO:1), EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY (SEQ ID NO: 3), andEGPWLEEEEEAYGWMDF (SEQ ID NO: 4).

In some embodiments, administration of a pharmaceutical composition ofthe presently disclosed subject matter to a subject induces a reductionin and/or prevents the development of fibrosis associated with thepancreatic cancer.

In some embodiments, the presently disclosed treatment methods aredesigned to inhibit growth and/or survival of a gastrin-associated tumorand/or cancer in a subject. In some embodiments, the presently disclosedmethods thus comprise administering to the subject a composition thatcomprises a first agent comprising a gastrin immunogen, one or moreanti-gastrin antibodies, one or more anti-CCK-B receptor antibodies, orany combination thereof; and a second agent comprising a checkpointinhibitor.

Thus, in some embodiments the presently disclosed subject matterprovides uses of the pharmaceutical compositions disclosed herein forthe preparation of medicaments to treat gastrin-associated tumors and/orcancers as well as uses of the pharmaceutical compositions disclosedherein to treat gastrin-associated tumors and/or cancers.

In some embodiments, the multi-agent pharmaceutical compositionsdisclosed herein provide enhanced, more efficacious, and/or moresuccessful treatment of gastrin-associated tumors and/or cancers thanwould treating a similar subject with the any of the agentsindividually.

VI.B. Methods for Inducing and/or Enhancing Cellular Immune ResponsesAgainst Gastrin-associated Tumors and/or Cancers

The presently disclosed subject matter also provides methods forinducing and/or enhancing cellular immune responses againstgastrin-associated tumors and/or cancers in subject. In someembodiments, the methods comprise administering to a subject that has agastrin-associated tumor or cancer an effective amount of a compositioncomprising an agent that reduces or inhibits gastrin signaling via CCK-Breceptors present on a gastrin-associated tumor or cancer, therebyinducing and/or enhancing a cellular immune response against thesubject's gastrin-associated tumor and/or cancer. As used herein, thephrase “inducing and/or enhancing a cellular immune response against agastrin-associated tumor and/or cancer” and grammatical variants ofrefers to a circumstance where as a result of administering to a subjectthat has a gastrin-associated tumor or cancer an effective amount of acomposition comprising an agent that reduces or inhibits gastrinsignaling via CCK-B receptors present on a gastrin-associated tumor orcancer, a level of a T cell-based immune response is higher in thesubject at a relevant time post-administration than would have beenpresent in the subject in the absence of the treatment. Agents thatreduce or inhibit gastrin signaling via CCK-B receptors present on agastrin-associated tumor or cancer include the agents disclosed hereinthat can interfere with an interaction of a gastrin peptide and a CCK-Breceptor, and include but are not limited to gastrin peptides and/orimmunogens, anti-gastrin antibodies, anti-CCK-B receptor antibodies,small molecule inhibitors of gastrin/CCK-B signaling, and combinationsthereof.

VI.C. Methods for Sensitizing Tumors and/or Cancers to Inducers ofCellular Immune Responses

In some embodiments, the presently disclosed subject matter alsoprovides methods for sensitizing tumors and/or cancers associated withgastrin and/or CCK-B receptor signaling in a subject to inducers ofcellular immune responses directed against the tumors and/or cancers. Asused herein, the phrase “sensitizing tumors and/or cancers associatedwith gastrin and/or CCK-B receptor signaling in a subject to inducers ofcellular immune responses” refers to treatments that result in levels ofcellular immune responses in subjects when one or more inducers of acellular immune response is administered to the subject as compared tolevels of cellular immune responses in subjects when one or moreinducers of a cellular immune response is administered to the subject inthe absence of the treatment.

In some embodiments, the methods comprise administering to a subject acomposition comprising a first agent that induces and/or provides anactive and/or a passive humoral immune response against a gastrinpeptide, and a second agent that induces and/or provides a cellularimmune response against the tumor and/or the cancer, or a combinationthereof, optionally wherein the first agent and the second agent areindividually selected from the group consisting of a gastrin peptideand/or a fragment and/or a derivative thereof that induces a cellularimmune response or production of neutralizing anti-gastrin antibodies inthe subject and a neutralizing anti-gastrin antibody and/or a fragmentand/or derivative thereof and; and/or a composition comprising a nucleicacid that inhibits expression of a gastrin gene product; and/or acomposition comprising an agent that blocks the biological function ofgastrin at the CCK-B receptor. In some embodiments, the anti-gastrinantibody is an antibody directed against an epitope present withingastrin-17 (G17).

Accordingly, in some embodiments the instant methods for sensitizingtumors and/or cancers associated with gastrin and/or CCK-B receptorsignaling in a subject to inducers of cellular immune responsescomprises administering to the subject a pharmaceutical composition asdisclosed herein in order to induce and/or provide to the subject bothan active and/or a passive humoral immune response against a gastrinpeptide in the subject as well as to induce and/or provide a cellularimmune response against the tumor and/or the cancer.

VI.D. Methods for Preventing, Reducing, and/or Eliminating FibrosisAssociated with Tumors and/or Cancers

PC is also characterized by a dense fibrotic environment (Neesse et al.,2011), which helps promote angiogenesis and creates a physical barrierthat could inhibit the penetration of chemotherapeutics andimmunotherapeutics to the pancreatic tumor site (Templeton & Brentnall,2013). Disclosed herein is the unexpected and surprising observationthat that with PAS administration, optionally in combination with one ormore immune checkpoint inhibitors, the fibrotic nature that is ahallmark of PC fibrosis can be reduced. While not wishing to be bound byany particular theory of operation, a reduction in fibrosis canfacilitate greater penetration of other drugs, including but not limitedto macromolecules like checkpoint mAbs. This could explain why checkpoint inhibitors have to date been characterized by very modestefficacy, perhaps due to lack of penetration of the checkpoint mAbs toPDAC cells. Therefore, an aspect of the presently disclosed subjectmatter is that PAS plus immune checkpoint inhibitors haveanti-PDAC-tumor activity separately when given as monotherapy, but whengiven as a combination therapy as disclosed herein, they have muchgreater activity.

Novel and innovative drug combinations with diverse but complementary oreven synergistic mechanisms of action are provided in accordance withthe presently disclosed subject matter to address the inherentlyfibrotic nature of PDAC and to be beneficial to allow greater access tothe tumor environment of large monoclonal antibodies (mAbs), such as butnot limited to anti-immune checkpoint inhibitor mAbs. While not wishingto be bound by any particular theory of operation, PAS plus immunecheckpoint inhibitors when administered together as part of acombination therapy can provide a synergistic effect to make tumors moreaccessible to chemotherapeutics and immune checkpoint inhibitor drugs byreducing the fibrosis associated with PDAC, thereby allowing anti-tumortherapeutics to target the interaction of PD-1 and PD-L1 in order toinduce a cellular immune response against a gastrin-associated tumor.

Treatment with PAS results in a humoral immunological response (i.e., anantibody response) to the autocrine and paracrine tumor/cancer growthfactor gastrin. In so doing, PAS affects the tumor/cancer (e.g., PDAC)phenotype by affecting cell proliferation, apoptosis, angiogenesis,invasion, and metastasis. As disclosed herein, PAS is also effective indecreasing fibrosis associated with PDAC. While not wishing to be boundby any particular theory of operation, this is believed to enhance theability of large molecules, such as but not limited to immune checkpointinhibitory mAbs, to gain greater access to the pancreatic tumor site,which in turn would be expected to promote a much greater cellularimmune effect. PAS also results in a cellular immune response togastrin. Thus, disclosed herein are methods for treating tumors and/orcancers by PAS administration in conjunction with the administration ofimmune checkpoint inhibitors such as anti-PD-1, anti-PD-L1, and/oranti-CTLA-4 mAbs to address the inherent fibrotic as well asrecalcitrant nature of PDAC in resistance to therapeutic agents thatneed access to the tumor for efficacy.

Therefore, in some embodiments the presently disclosed subject matterprovides methods for preventing, reducing, and/or eliminating formationof fibrosis associated with a tumor and/or a cancer, optionallypancreatic cancer, by contacting cells of the tumor and/or the cancerwith an agent that directly or indirectly inhibits one or morebiological activities of gastrin in the tumor and/or cancer. Agents thatdirectly or indirectly inhibit one or more biological activities ofgastrin are disclosed herein above, and include agents that provideand/or induce humoral immune responses against gastrin peptides (such asbut not limited to anti-gastrin antibodies, and/or fragments and/orderivatives thereof), and gastrin peptides that induce production ofneutralizing anti-gastrin antibodies in the subject; inhibitory nucleicacids that inhibit expression of gastrin gene products; small moleculecompounds that block the function of the gastrin hormone, and anycombination thereof. In some embodiments, the anti-gastrin antibodiescomprise an antibody directed against an epitope present withingastrin-17 (G17), which epitope is in some embodiments present withinone or more of the amino acid sequences EGPWLEEEEE (SEQ ID NO: 1),EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY (SEQ ID NO: 3), andEGPWLEEEEEAYGWMDF (SEQ ID NO: 4).

As with other immunogenic forms of gastrin and gastrin peptidesdisclosed herein, in some embodiments the gastrin peptides areconjugated to an immunogenic carrier, optionally an immunogenic carrierselected from the group consisting of diphtheria toxoid, tetanus toxoid,keyhole limpet hemocyanin, and bovine serum albumin.

In some embodiments, the methods for preventing, reducing, and/oreliminating formation of fibrosis associated with a tumor and/or acancer, optionally pancreatic cancer further comprise contacting thetumor and/or the cancer with a second agent comprising a stimulator of acellular immune response against the tumor and/or the cancer. Exemplarystimulators of cellular immune responses include immune checkpointinhibitors such as those that inhibit a biological activity of a targetpolypeptide selected from the group consisting of cytotoxic T-lymphocyteantigen 4 (CTLA4), programmed cell death-1 receptor (PD-1), andprogrammed cell death 1 receptor ligand (PD-L1), including but notlimited to

Ipilimumab, Tremelimumab, Nivolumab, Pidilizumab, Pembrolizumab, AMP514,AUNP12, BMS-936559/MDX-1105, Atezolizumab, MPDL3280A, RG7446, R05541267,MEDI4736, and Avelumab.

In some embodiments, the tumor and/or cancer for which preventing,reducing, and/or eliminating the formation of fibrosis therein ispancreatic cancer.

VI.E. Methods for Modulating T Cell Subpopulations in Subjects andTumors Present Therein

As disclosed herein, administration of the pharmaceutical compositionsof the presently disclosed subject matter to subjects that havegastrin-associated tumors and/or cancers was observed to modify both thecirculating T cell subpopulations present in subjects treated withgastrin-associated tumors and/or cancers as well as the T cellsubpopulations present within the tumors and/or cancers per se.

In some embodiments, administration of the pharmaceutical compositionsof the presently disclosed subject matter to subjects that havegastrin-associated tumors and/or cancers results in an enhancement ofthe number of CD8⁺ tumor infiltrating lymphocytes (TILs) present ingastrin-associated tumors and/or cancers. It is recognized in the artthat that TILs have anti-tumor and anti-cancer activity, and thusincreasing the number of TILs in a tumor and/or a cancer can result ingreater anti-tumor and/or anti-cancer efficacy of various treatmentstrategies with either the pharmaceutical compositions of the presentlydisclosed subject matter alone or in combination with other front-linean/d or secondary treatments.

In some embodiments, administration of the pharmaceutical compositionsof the presently disclosed subject matter to subjects that havegastrin-associated tumors and/or cancers results in a reduction in thenumber of FoxP3⁺ inhibitory T-regulatory cells (T_(regs)) present ingastrin-associated tumors and/or cancers. It is recognized in the artthat that T_(regs) have immunosuppressive activity, particularly tumor-and cancer-specific immunosuppressive activity, and thus reducing thenumber of FoxP3⁺ inhibitory T_(regs) in a tumor and/or a cancer canresult in greater anti-tumor and/or anti-cancer efficacy of varioustreatment strategies with either the pharmaceutical compositions of thepresently disclosed subject matter alone or in combination with otherfront-line an/d or secondary treatments. In some embodiments, reducingthe number of FoxP3⁺ inhibitory T_(regs) in a tumor and/or a cancer canresult in greater efficacy of front-line chemotherapeutics.

In some embodiments, administration of the pharmaceutical compositionsof the presently disclosed subject matter to subjects that havegastrin-associated tumors and/or cancers results in an increase inanti-gastrin T_(EMRA) cells in the subjects. T_(EMRA) cells are effectormemory T cells that are found in the peripheral circulation and tissues.T_(EMRA) cells appear to have a sentinel activity in that they might beinvolved in recognizing metastases. As such, increasing anti-gastrinT_(EMRA) cells in subjects could prevent, reduce, and/or eliminatemetastasis associated with gastrin-associated tumors and/or cancers.Therefore, in some embodiments the presently disclosed subject matterrelates to methods for increasing T_(EMRA) cells that recognizegastrin-associated tumor and/or cancer antigens and cells expressing thesame by treating subjects with the pharmaceutical compositions disclosedherein.

Summarily, in some embodiments the presently disclosed subject matterrelates to uses of the presently disclosed compositions comprisingimmune checkpoint inhibitors and gastrin immunogens to treatgastrin-associated tumors and/or cancers, either alone as a front-linetherapy, in combination with other front-line therapies, or incombination with any other therapy that would be appropriate for asubject who has a gastrin-associated tumor and/or cancer.

VII. CONCLUSION

The presently disclosed subject matter thus relates in some embodimentsto combination therapies for the treatment of cancer using a combinationof methods that individually or together generate both a humoralantibody immune response (using, for example, the gastrin cancer vaccinePAS) and a cellular T cell immune response (using, for example, thegastrin cancer vaccine PAS or an immune checkpoint inhibitor). Moreparticularly, unexpected additive and/or synergistic efficacies intreating human and animal gastrointestinal tumors using the instantlydescribed combination of drug classes that generate humoral and cellularimmune anti-tumor responses in combination with cellular immuneanti-tumor effects are described.

More particularly, the presently disclosed subject matter relates insome embodiments to using specific combinations of drugs that (i) inducehumoral B cell immune responses to a tumor growth factor or circulatingtumor growth factor; and (ii) induce and/or enhance cellular immuneresponses (i.e., anti-tumor and/or cancer T cell responses) directedagainst the tumor and/or cancer to elicit a cytotoxic T lymphocyteresponse.

As such, in some embodiments disclosed herein are methods for treatinghuman and animal tumors and cancers using a combination of a gastrincancer vaccine in combination with a second drug that overcomes immunecheckpoint failure. Thus, in some embodiments the presently disclosedsubject matter relates to treating specific human cancers with a cancervaccine directed at eliciting a B cell and/or antibody immune responseand a cellular immune response to the active form of the growth factorgastrin, with the unexpected observation that this vaccine treatmentalso resulted in making the tumor more responsive to treatment with animmune checkpoint inhibitor, thus creating an unexpected, additive, oreven synergistic combination therapeutic effect that enhanced anti-tumorefficacy.

Additionally, the pharmaceutical compositions of the presently disclosedsubject matter can be employed for preventing, reducing, and/oreliminating metastasis of a gastrin-associated tumor or cancer byadministering to a subject having a gastrin-associated tumor or canceran amount of the pharmaceutical composition of any one of claims 1-13sufficient to enhance the number of CD8+ tumor infiltrating lymphocytes.

The use of claim 104, wherein the administering results in improvessurvival of the subject, reduced tumor growth, and/or enhanced efficacyof a chemotherapeutic agent and/or an immune checkpoint therapy in thesubject as compared to that which would have occurred had thepharmaceutical composition not been administered.

Use of the pharmaceutical composition of any one of claims 1-13 forpreventing, reducing, and/or eliminating metastasis of agastrin-associated tumor or cancer by administering to a subject havinga gastrin-associated tumor or cancer an amount of the pharmaceuticalcomposition of any one of claims 1-13 sufficient to reduce the number ofFoxP3+ inhibitory T-regulatory cells.

EXAMPLES

The following EXAMPLES provide illustrative embodiments. In light of thepresent disclosure and the general level of skill in the art, those ofskill will appreciate that the following EXAMPLES are intended to beexemplary only and that numerous changes, modifications, and alterationscan be employed without departing from the scope of the presentlydisclosed subject matter.

Materials and Methods for the EXAMPLES

Cell Line:

Murine mT3 pancreatic cancer cells were obtained from the laboratory ofDr. David Tuveson (Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y., United States of America; see also Boj et al., 2015). These cellshave been shown to express the CCK-B receptor and produce gastrin, andwere used as the tumor model. These cells produce tumors in syngeneicC57BL/6 mice (Smith et al., 2018).

Study Design:

All animal studies were performed in an ethical fashion under a protocolapproved by the Institutional Animal Care and Use Committee (IACUC) ofGeorgetown University (Washington, D.C., United States of America).Forty male (6 weeks old) C57BL-6 mice were injected with 500,000 cellssubcutaneously into the flank. On the 6^(th) day after inoculation 100%of the mice had a palpable tumor and were allocated into one of four (4)groups of n=10 mice each so that the baseline tumor volume was equal inall groups. The Groups were as follows:

1. PBS Control (PBS)

2. PAS 100 μg (PAS100)

3. PD-1 Ab 150 μg (PD-1)

4. PD-1 Ab (150 μg)+PAS 100 μg (PD-1+PAS100)

One week (7 days) after the mT3 cells were injected, non-control Groupsof mice received administration of patent applications and/or PD-1 Ab asfollows: if the mice were in a group that was to receive PAS, the PASwas injected starting at the time of randomization (baseline time=0) asan i.p. injection in 100 μl and again at week 1 and at week 3. PD-1antibody (Bio X cell, West Lebanon, N.H., United States of America) wasgiven to appropriate mice at a dose of 150 μg i.p. five times during thestudy at t=0, 4, 8, 15, and 21 days). Control mice received PBS on thesame days that PAS was administered. Tumor volumes were measured weeklyby calipers and calculated as L×(w)²×0.5.

Histology:

After 31 days of growth the mice were ethically euthanized by CO₂asphyxiation and cervical dislocation. Mice were weighed, pancreatictumors were excised, and they were weighed. The tumors were divided andhalf of the tumor was fixed in 4% paraffin in formaldehyde for histologyand half was flash frozen in liquid nitrogen. Tumor-associated fibrosiswas assessed with Masson's trichrome staining. Analysis of Masson'strichrome was done by a technician blinded to the treatment using ImageJimage processing and analysis software (developed by Wayne Rasband ofthe United States National Institutes of Health (NIH), Bethesda, Md.,United States of America; available through the website of the NIH).

For immunohistochemistry, tumors were sectioned from paraffin embeddedblocks (10 μm) and fixed on slides. Tumor sections were stained witheither anti-CD8 antibodies (1:75; EBIOSCIENCE™, San Diego, Calif.,United States of America); or anti-Foxp3 antibodies (1:30;EBIOSCIENCE™). Immunoreactive cells were counted manually.

Spleen T-Cell Isolation.

The spleen from each animal was removed, weighed, and placed in a 60 mmdish containing 5 ml RPMI1640 medium. The spleens were mechanicallychopped using a razor blade. The medium containing the spleen tissue wasfiltered through a 100 μM cell strainer to a 50 ml tube and rinsed withmedium a few times until the final volume was 40 ml. The spleen tissuewas then filtered again using a 40 μM cell strainer to a 50 ml tube, andcentrifuged to pellet down the cells at 1500 rpm for 5 minutes at 4° C.The supernatant was removed and the cell pellet resuspended in 40 ml PBSbefore the cells were repelleted by centrifugation at 1500 rpm for 5minutes at 4° C. The supernatant was discarded, the cell pellet wasresuspended in 3 ml Washing buffer (PBS with 2 mM EDTA and 0.5% bovineserum albumin), and then slowly added on the top of 5 ml Ficoll mediumin a 15 ml tube. After centrifugation at 2100 rpm for 20 minutes withdeceleration set to zero, the lymphocytes were collected from the whitelayer between buffer and the Ficoll. The lymphocytes were washed anadditional two times, resuspended in medium, and counted.

Flow Cytometry.

One million lymphocytes were added to a 5 ml clear tube (Catalogue#352054; BD Falcon, Bedford, Mass., United States of America), volumeswere equalized with PBS, and the cells were pelleted at 1500 rpm for 5minutes. After washing with PBS, 50 μl of pre-diluted ZOMBIE NIR™ brandfixable viability solution (BIOLEGEND®, San Diego, Calif., United Statesof America) was added to the cells, which were then incubated at roomtemperature in the dark for 20 minutes. The cells were washed and thenblocked by adding 5 μl Purified Rat Anti-Mouse CD16/CD32 (Mouse BD FcBLOCK™ brand reagent; BD Biosciences, San Jose, Calif., United States ofAmerica) and incubating for 20 minutes.

The antibodies listed in Table 1 were reacted to the lymphocytes andflow cytometry performed using a FACSARIA™ Ilu brand cell sorter (BDBiosciences) with 375 nm, 405 nm, 488 nm and 633 nm laser lines.

TABLE 1 Antibodies Employed for T-cell Staining for Flow CytometryFluorescent Label Antigen Supplier PE CD4 EBIOSCIENCE ™ (San Diego,California, United States of America) Fitc CD3 BIOLEGEND ® (San Diego,California, United States of America) PE/Dazzle 594 CD62L BIOLEGEND ®eFlour 450/BV421 CD8a EBIOSCIENCE ™ APC CD25 BIOLEGEND ® BV 605 CD69BIOLEGEND ® BV 510 CD44 BIOLEGEND ® BV 650 CD45 BIOLEGEND ®

For Re-Stimulation.

1 or 2 million isolated and washed lymphocytes were added to each wellof a 6-well plate for two duplicate plates, and the volume was broughtto the same for each (2 or 3 ml). Brefeldin A solution (BIOLEGEND®,1000X Catalogue No. 420601) was added at 1 μl/ml to each well. 1 μMgastrin-14 (Sigma Aldrich Catalogue No. SCP0152, having the amino acidsequence pEGPWLEEEEEAYGW; SEQ ID NO: 5) was added to each well at 1μl/ml for one plate for a final gastrin concentration of 1 nM. The otherduplicate plate was not treated with gastrin-14 and served as a control.The 6-well plates were placed in the cell culture incubator at 37° C.for 6 hours. The cells were then removed, washed, and permeabilizedusing an Intracellular Fixation & Permeabilization Buffer Set(EBIOSCIENCE™ Catalogue No. 88-8824-00). A cytokine antibody master mixincluding the four (4) antibodies listed in Table 2 was added (4antibodies for 8 samples, so 10 μl of each antibody to make themastermix), and incubated at 4° C. overnight.

TABLE 2 Antibodies Employed for Re-stimulation Analyses FluorescentLabel Target Supplier PE/Dazzle594 TNFα EBIOSCIENCE ™ APC IFNγEBIOSCIENCE ™ PE Granzyme-B EBIOSCIENCE ™ FITC Perforin EBIOSCIENCE ™

Flow cytometry was performed to analyze for cytokines in cells that werere-stimulated with gastrin or with PBS. Analysis of flow cytometry datawas done using FCSExpress-6 software (De Novo Software, Glendale,Calif., United States of America).

Example 1 Producing Tumors in Mice

To determine whether PAS treatment induced both a humoral and a cellularimmune response and provided a synergistic effect on immune checkpointantibody therapy, tumors were generated in immune competent mice (e.g.,C57BL/6 mice that were syngeneic with murine mT3 pancreatic cancercells) by introducing 5×10⁵ murine mT3 pancreatic cancer cells in 0.1 mlPBS subcutaneously into the flank. After allowing for one week for thetumors to become established, mice were treated with PAS and one or moreimmune checkpoint inhibitors as depicted in FIG. 1.

Animals were treated starting one week after mT3 pancreatic cancer cellinoculation as this timeframe ensured that all animals in the study hada palpable subcutaneous tumor, and that treatment did not interfere withtumor initiation. The primary end points were tumor growth and survival.Growth of tumors was measured weekly with calipers and the volumes ofthe tumors were calculated as L×W²×0.5. Tumors were excised and examinedhistologically by immunohistochemistry for immune cells, including butnot limited to tumor-infiltrating lymphocytes (TILs), and T Regulatorycells (T_(regs)). Tumors were also examined for the presence and extentof fibrosis development typically associated with PDAC. Spleens wereremoved and T-cells isolated and re-stimulated with gastrin. The cellswere labeled with a panel of relevant antibodies for cytokines andcharacterized by flow cytometry.

Each experiment employed 40 mice (n=10 per group; see FIG. 1), whichwere implanted with 5×10⁵ pancreatic murine cancer cells. Groups ofimmune competent syngeneic mice bearing mT3 murine pancreatic tumorswere treated with PBS (negative control), PAS monotherapy (100 μg peradministration at 0, 1, 2, and 3 weeks after tumor cell inoculation), ananti-PD-1 antibody (PD1-1 Ab; Bio X cell, West Lebanon, N.H., UnitedStates of America) as an immune checkpoint inhibitor (150 μg peradministration at 0, 4, 8, 15, and 21 days after the first PASvaccination), or a combination of both PAS vaccination (100 μg peradministration at 0, 1, 2, and 3 weeks after tumor cell inoculation) andthe immune checkpoint inhibitor (150 μg per administration at 0, 4, 8,15, and 21 days after the first PAS vaccination). The immune checkpointblockade antibody specific for programmed cell death protein 1 (PD1-1Ab; Bio X cell, West Lebanon, N.H., United States of America) wasadministered intraperitoneally. The data are summarized in Table 3 belowand in FIG. 2.

TABLE 3 Mean Mouse Body Weight in Each Treatment Group Treatment GroupMean Weight (g ± SEM) p value PBS (negative control) 27.500 ± 0.563 —PD-1 27.333 ± 0.645 NS PAS100 alone 27.400 ± 0.499 NS PD-1 + PAS10028.000 ± 0.577 NS NS: not significant

There were no statistical differences in final tumor weights in gramsbetween the PBS control mice and the weights of the tumors from mice inboth the PD-1 and PAS100-treated groups. In contrast, the mice treatedwith the combination of PD-1 and PAS100 had significantly smaller tumorscompared to PBS (p=0.014) and PD-1 controls (p=0.0017). Furthermore, thecombination therapy with PD-1 and PAS100 resulted in tumors that werealso significantly smaller than PAS100 monotherapy (p<0.05).

Example 2 Analyses of T_(EMRA) CD4⁻/CD8⁻ Cells in the CD3 TerminallyDifferentiated T Cell Subpopulation

Tumors were induced in mice as set forth in EXAMPLE 1. T lymphocyteswere isolated from spleen peripheral blood mononuclear cells (PBMCs)that were isolated from mice that had been treated with PBS, PD-1 Ab,PAS100, or PAS100+PD-1 Ab. Various subpopulations of T cells wereidentified by flow cytometry using the antibodies listed in Table 1. Inparticular, a first T cell subpopulation was isolated that wasCD3⁺/CD4⁻/CD8⁻, and from this subpopulation a further subpopulationrepresenting T_(EMRA) cells that were CD3⁺/CD4⁻/CD8⁻/CD44⁻/CD62L⁻ wasisolated. The percentages and proportions of these varioussubpopulations present in mice that had been treated with PBS, PD-1 Ab,PAS100, or PAS100+PD-1 Ab were determined, and the results are presentedin FIGS. 3A and 3B.

FIG. 3A shows the percentage of T_(EMRA) cells(CD3⁺/CD4⁻/CD8⁻/CD44⁻/CD62L⁻) in CD3⁺ T cells in mice treated with PBS,PD-1 Ab, PAS100, or PAS100/PD-1. FIG. 3B shows the proportion ofCD3⁺/CD4⁻/CD8⁻ cells in each treatment group that were T_(EMRA) cells.

The most significant differences among the treatment groups were thatPAS100 had lower CD4⁻/CD8⁻ T_(EMRA) cells than that of PBS, whereasPAS100+PD-1 treatment resulted in similar CD4⁻/CD8⁻ T_(EMRA) cells ascompared to that of PBS. The portion of T_(EMRA) cells(CD3⁺/CD4⁻/CD8⁻/CD44⁻/CD62L⁻) in T cells from mice treated withPAS100/PD1 was over 2-fold higher than that from mice treated with PBSor PAS100 alone, suggesting that T_(EMRA) cells(CD3⁺/CD4⁻/CD8⁻/CD44⁻/CD62L⁻) were good for defending against andfighting gastrin-associated tumors and cancers.

Example 3 Cytokine Activation Assay with PAS100

T lymphocytes were isolated from spleen peripheral blood mononuclearcells (PBMCs) that were isolated from mice that had been treated withPAS100. These cells were evaluated by flow cytometry to determine ifthey were indeed activated T-cells by cytokine activation toInterferon-γ (INFG), Granzyme-B (granzyme), Perforin, and tumor necrosisfactor-α(TNFα). The results are provided in FIGS. 4A and 4B.

FIG. 4A shows that the T cells isolated from mice treated with PAS100were indeed activated. When these same cells were re-stimulated withgastrin in culture for 6 hours (see FIG. 4B), they were re-stimulatedand released more cytokines, confirming that vaccination with PAS100stimulated T cells and further that these T cells specifically reactedto gastrin.

Example 4 Comparison of PAS100 to Combination Therapy with PAS & PD-1

T lymphocytes were isolated from spleen PBMC isolated from mice that hadbeen treated with PAS100 or a combination of PAS100 and PD-1. Ccellswere evaluated by flow cytometry to determine if they were indeedactivated T cells by cytokine activation to Interferon-γ (INFG),Granzyme-B (granzyme), Perforin, and tumor necrosis factor-α (TNFa). Theresults are provided in FIGS. 5A and 5B.

Activated T lymphocytes from mice treated with PAS100 alone releasedincreased cytokines compared to lymphocytes from PBS treated mice (seeFIG. 5A). The lymphocytes from the combination treated mice, however,released markedly more cytokines (see FIG. 5B), suggesting that thecombination therapy was better at stimulating activated T cells. TNFα inparticular was increased greater than 2-fold with the PAS100+PD-1 Abcombination therapy as compared to treatment with PAS100 alone (compareFIGS. 5A and 5B).

Example 5 Analysis of the Effect of PD-1 Monotherapy, PAS100Monotherapy, and PD-1+PAS100 Combination Therapy on Fibrosis

Tumors from mice treated with PBS, PD-1 alone, PAS100, or PAS100+PD-1were fixed in 4% paraformaldehyde, paraffin embedded, and 8 μm sectionswere cut and mounted. Tissue sections were stained for fibrosis withMasson's trichrome.

Representative sections stained with Masson's trichrome are shown inFIG. 6A. Fibrosis quantitative scores were analyzed by a computerprogram using ImageJ image processing and analysis software, and theresults are presented in FIG. 6B. Of note is that whereas the integrateddensity of the tumors treated with PD-1 monotherapy and PAS100monotherapy were insignificantly different the negative control PBStreatment, the PAS+PD-1 Ab combination therapy resulted in a decrease indensity (and hence fibrosis) that was statistically significant ascompared to PBS alone (p<0.005) and also PAS100 alone (p<0.001).

Example 6 Analysis of the Effect of PD-1 Monotherapy, PAS100Monotherapy, and PD-1+PAS100 Combination Therapy on CD8⁺ T CellInfiltration

Tumors were fixed in 4% paraformaldehyde, paraffin embedded, and 8 μmsections were cut and mounted. CD8⁺ lymphocytes were stained in thetumor microenvironment with CD8 antibodies (1:75 titer; EBIOSCIENCE™,San Diego, Calif., U1SA) and CD8⁺ cells were manually counted in ablinded fashion. The results are presented in FIGS. 7A and 7B.

As shown in FIGS. 7A and 7B, CD8⁺ tumor-infiltrating lymphocytes (TILs)increased with PAS100 and PD-1 alone, but were markedly increased withthe combination therapy. The combination PAS100+PD-1 CD8⁺ cells weresignificantly greater than PD-1 alone (p=0.042) and greater than PAS100alone (p=0.039).

Example 7 Analysis of the Effect of PD-1 Monotherapy, PAS100Monotherapy, and PD-1+PAS100 Combination Therapy on Foxp3⁺ T_(reg)Infiltration

Tumors were fixed in 4% paraformaldehyde, paraffin embedded, and 8 μmsections were cut and mounted. Tumors were reacted with an anti-Foxp3antibody (1:30; EBIOSCIENCE™) and immunoreactive cells counted manuallyusing ImageJ software. The results are presented in FIGS. 8A and 8B.

FIG. 8A depicts exemplary mT3 tumors stained with an antibody that bindsto the Foxp3 protein, a marker for T_(regs). Comparison of the fieldsshows that as compared to PBS (upper left panel), PD-1 monotherapy(upper right panel), or PAS100 monotherapy (lower left panel), PAS100 &PD-1 combination therapy resulted in a decrease in the presence ofintratumoral T_(regs), suggesting that PAS100+PD-1 combination therapymight modify the intratumoral environment to an extent where theintratumoral microenvironment might be characterized by a lower degreeof T_(reg)-based immunosuppression as compared to either monotherapyalone.

FIG. 8B is a bar graph summarizing the data exemplified by FIG. 8A. Ascompared to PBS, the number of Foxp3⁺ cells in tumors treated with PD-1monotherapy or PAS100 monotherapy was not significantly different.Tumors treated with PAS100+PD-1 combination therapy had significantlyfewer Foxp3⁺ cells that the negative control.

REFERENCES

All references listed in the instant disclosure, including but notlimited to all patents, patent applications and publications thereof,scientific journal articles, and database entries (including but notlimited to GENBANK® biosequence database entries and including allannotations available therein) are incorporated herein by reference intheir entireties to the extent that they supplement, explain, provide abackground for, and/or teach methodology, techniques, and/orcompositions employed herein. The discussion of the references isintended merely to summarize the assertions made by their authors. Noadmission is made that any reference (or a portion of any reference) isrelevant prior art. Applicants reserve the right to challenge theaccuracy and pertinence of any cited reference.

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It will be understood that various details of the presently disclosedsubject matter may be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

What is claimed is:
 1. A pharmaceutical composition comprising (i) afirst agent that induces and/or provides an active and/or a passivehumoral immune response against a gastrin peptide and/or a CCK-Breceptor; and (ii) an immune checkpoint inhibitor.
 2. The pharmaceuticalcomposition of claim 1, wherein the first agent is selected from thegroup consisting of a gastrin peptide, an anti-gastrin antibody, and ananti-CCK-R antibody.
 3. The pharmaceutical composition of claim 2,wherein the first agent comprises a gastrin peptide, optionally agastrin peptide comprising, consisting essentially of, or consisting ofan amino acid sequence selected from the group consisting of EGPWLEEEEE(SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY (SEQ ID NO: 3),and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4).
 4. The pharmaceutical compositionof claim 3, wherein the gastrin peptide is conjugated to an immunogeniccarrier, optionally via a linker.
 5. The pharmaceutical composition ofclaim 4, wherein the immunogenic carrier is selected from the groupconsisting of diphtheria toxoid, tetanus toxoid, keyhole limpethemocyanin, and bovine serum albumin.
 6. The pharmaceutical compositionof claim 4, wherein the linker comprises a ε-maleimido caproic acidN-hydroxysuccinamide ester.
 7. The pharmaceutical composition of claim 4or claim 6, wherein the linker and the gastrin peptide are separated byan amino acid spacer, optionally wherein the amino acid spacer isbetween 1 and 10 amino acids in length, further optionally wherein theamino acid spacer is 7 amino acids in length.
 8. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutical composition furthercomprises an adjuvant, optionally an oil-based adjuvant.
 9. Thepharmaceutical composition of claim 1, wherein the immune checkpointinhibitor inhibits a biological activity of a target polypeptideselected from the group consisting of cytotoxic T-lymphocyte antigen 4(CTLA4), programmed cell death-1 receptor (PD-1), and programmed celldeath 1 receptor ligand (PD-L1).
 10. The pharmaceutical composition ofclaim 1, wherein the immune checkpoint inhibitor is selected from thegroup consisting of Ipilimumab, Tremelimumab, Nivolumab, Pidilizumab,Pembrolizumab, AMP514, AUNP12, BMS-936559/MDX-1105, Atezolizumab,MPDL3280A, RG7446, R05541267, MEDI4736, Avelumab and Durvalumab.
 11. Thepharmaceutical composition of claim 1, wherein the gastrin-associatedtumor and/or cancer is pancreatic cancer.
 12. The pharmaceuticalcomposition of claim 1, wherein the first agent comprises an amount of agastrin peptide comprising, consisting essentially of, or consisting ofan amino acid sequence selected from the group consisting of EGPWLEEEEE(SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY (SEQ ID NO: 3),and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4) effective to induce an anti-gastrinhumoral response and the second agent comprises an amount of an immunecheckpoint inhibitor that is effective to induce or enhance a cellularimmune response against a gastrin-associated tumor or cancer whenadministered to a subject who has gastrin-associated tumor or cancer.13. The pharmaceutical composition of claim 1, wherein the first agentcomprises one or more anti-CCK-B receptor antibodies and is present inthe pharmaceutical composition in an amount sufficient to reduce orinhibit gastrin signaling via CCK-B receptors present on agastrin-associated tumor or cancer when administered to a subject thathas a gastrin-associated tumor or cancer.
 14. The pharmaceuticalcomposition of any one of claims 1-13 for use in producing a medicamentfor treating a gastrin-associated tumor or cancer.
 15. Thepharmaceutical composition of any one of claims 1-13 for use in treatinga gastrin-associated tumor or cancer.
 16. A method for treating agastrin-associated tumor or cancer in a subject, the method comprisingadministering to the subject an effective amount of a composition thatcomprises (i) a first agent that induces and/or provides an activeand/or a passive humoral immune response against a gastrin peptideand/or a CCK-B receptor; and (ii) a second agent that induces and/orprovides a cellular immune response against the gastrin-associated tumoror cancer.
 17. The method of claim 16, wherein the first agent isselected from the group consisting of a gastrin peptide, an anti-gastrinantibody, and an anti-CCK-R antibody.
 18. The method of claim 16,wherein the first agent comprises a gastrin peptide, optionally agastrin peptide comprising, consisting essentially of, or consisting ofan amino acid sequence selected from the group consisting of EGPWLEEEEE(SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY (SEQ ID NO: 3),and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4).
 19. The method of claim 18,wherein the gastrin peptide is conjugated to an immunogenic carrier,optionally via a linker.
 20. The method of claim 19, wherein theimmunogenic carrier is selected from the group consisting of diphtheriatoxoid, tetanus toxoid, keyhole limpet hemocyanin, and bovine serumalbumin.
 21. The method of claim 19, wherein the linker comprises aε-maleimido caproic acid N-hydroxysuccinamide ester.
 22. The method ofclaim 19 or claim 21, wherein the linker and the gastrin peptide areseparated by an amino acid spacer, optionally wherein the amino acidspacer is between 1 and 10 amino acids in length, further optionallywherein the amino acid spacer is 7 amino acids in length.
 23. The methodof claim 16, wherein the composition further comprises an adjuvant,optionally an oil-based adjuvant.
 24. The method of claim 16, whereinthe inducer of the cellular immune response against thegastrin-associated tumor or cancer comprises an immune checkpointinhibitor.
 25. The method of claim 24, wherein the immune checkpointinhibitor inhibits a biological activity of a target polypeptideselected from the group consisting of cytotoxic T-lymphocyte antigen 4(CTLA4), programmed cell death-1 receptor (PD-1), and programmed celldeath 1 receptor ligand (PD-L1).
 26. The method of claim 25, wherein theimmune checkpoint inhibitor is selected from the group consisting ofIpilimumab, Tremelimumab, Nivolumab, Pidilizumab, Pembrolizumab, AMP514,AUNP12, BMS-936559/MDX-1105, Atezolizumab, MPDL3280A, RG7446, R05541267,MEDI4736, Avelumab and Durvalumab.
 27. The method of claim 16, whereinthe gastrin-associated tumor and/or cancer is pancreatic cancer.
 28. Themethod of claim 27, wherein the composition induces a reduction inand/or prevents the development of fibrosis associated with thepancreatic cancer.
 29. The method of claim 16, wherein the compositionis administered in a dose selected from the group consisting of about 50μg to about 1000 μg, about 50 μg to about 500 μg, about 100 μg to about1000 μg, about 200 μg to about 1000 μg, and about 250 μg to about 500μg, and optionally wherein the dose is repeated once, twice, or threetimes, optionally wherein the second dose is administered 1 week afterthe first dose and the third dose, if administered, is administered 1 or2 weeks after the second dose.
 30. A method for treating agastrin-associated tumor and/or cancer, the method comprisingadministering to a subject in need thereof a first agent that directlyor indirectly inhibits one or more biological activities of gastrin inthe tumor and/or cancer and a second agent comprising a stimulator of acellular immune response against the tumor and/or the cancer.
 31. Themethod of claim 30, wherein the first agent: (i) directly or indirectlyinhibits one or more biological activities of gastrin in the tumorand/or cancer by providing and/or inducing a humoral immune responseagainst a gastrin peptide, optionally wherein the agent is selected fromthe group consisting of an anti-gastrin antibody and a gastrin peptidethat induces production of neutralizing anti-gastrin antibodies in thesubject; and/or (ii) comprises a nucleic acid that inhibits expressionof a gastrin gene product.
 32. The method of claim 31, wherein theanti-gastrin antibody is an antibody directed against an epitope presentwithin gastrin-17 (G17).
 33. The method of claim 30, wherein the epitopeis present within the amino acid sequence EGPWLEEEEE (SEQ ID NO: 1),EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY (SEQ ID NO: 3), orEGPWLEEEEEAYGWMDF (SEQ ID NO: 4).
 34. The method of any one of claims30-33, wherein the first agent comprises the gastrin peptide conjugatedto an immunogenic carrier.
 35. The method of claim 34, wherein thegastrin peptide comprises an amino acid sequence selected from the groupconsisting of EGPWLEEEEE (SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2),EGPWLEEEEEAY (SEQ ID NO: 3), and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4). 36.The method of claim 34, wherein the immunogenic carrier is selected fromthe group consisting of diphtheria toxoid, tetanus toxoid, keyholelimpet hemocyanin, and bovine serum albumin.
 37. The method of claim 34,wherein the gastrin peptide is conjugated to the immunogenic carrier viaa linker.
 38. The method of claim 37, wherein the linker comprises aε-maleimido caproic acid N-hydroxysuccinamide ester.
 39. The method ofclaim 37 or claim 38, wherein the linker and the gastrin peptide areseparated by an amino acid spacer, optionally wherein the amino acidspacer is between 1 and 10 amino acids in length, further optionallywherein the amino acid spacer is 7 amino acids in length.
 40. The methodof any of claims 30-39, wherein the first agent further comprises anadjuvant, optionally an oil-based adjuvant.
 41. The method of any ofclaims 30-40, wherein the second agent is an immune checkpointinhibitor.
 42. The method of claim 41, wherein the immune checkpointinhibitor inhibits a biological activity of a target polypeptideselected from the group consisting of cytotoxic T-lymphocyte antigen 4(CTLA4), programmed cell death-1 receptor (PD-1), and programmed celldeath 1 receptor ligand (PD-L1).
 43. The method of claim 42, wherein theimmune checkpoint inhibitor is selected from the group consisting ofIpilimumab, Tremelimumab, Nivolumab, Pidilizumab, Pembrolizumab, AMP514,AUNP12, BMS-936559/MDX-1105, Atezolizumab, MPDL3280A, RG7446, R05541267,MEDI4736, and Avelumab.
 44. The method of any of claims 30-43, whereinthe gastrin-associated tumor and/or cancer is pancreatic cancer.
 45. Themethod of claim 44, wherein the first agent induces a reduction inand/or prevents the development of fibrosis associated with thepancreatic cancer.
 46. A method for inhibiting growth of agastrin-associated tumor and/or cancer in a subject, the methodcomprising administering to the subject a composition that comprises afirst agent comprising a gastrin immunogen, one or more anti-gastrinantibodies, one or more anti-CCK-B receptor antibodies, or anycombination thereof; and a second agent comprising an immune checkpointinhibitor.
 47. The method of claim 46, wherein the first agent isselected from the group consisting of a gastrin peptide, an anti-gastrinantibody, and an anti-CCK-R antibody.
 48. The method of claim 46,wherein the first agent comprises a gastrin peptide, optionally agastrin peptide comprising, consisting essentially of, or consisting ofan amino acid sequence selected from the group consisting of EGPWLEEEEE(SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY (SEQ ID NO: 3),and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4).
 49. The method of claim 48,wherein the gastrin peptide is conjugated to an immunogenic carrier,optionally via a linker.
 50. The method of claim 49, wherein theimmunogenic carrier is selected from the group consisting of diphtheriatoxoid, tetanus toxoid, keyhole limpet hemocyanin, and bovine serumalbumin.
 51. The method of claim 49, wherein the linker comprises aε-maleimido caproic acid N-hydroxysuccinamide ester.
 52. The method ofclaim 49 or claim 51, wherein the linker and the gastrin peptide areseparated by an amino acid spacer, optionally wherein the amino acidspacer is between 1 and 10 amino acids in length, further optionallywherein the amino acid spacer is 7 amino acids in length.
 53. The methodof claim 46, wherein the composition further comprises an adjuvant,optionally an oil-based adjuvant.
 54. The method of claim 46, whereinthe inducer of the cellular immune response against thegastrin-associated tumor or cancer comprises an immune checkpointinhibitor.
 55. The method of claim 54, wherein the immune checkpointinhibitor inhibits a biological activity of a target polypeptideselected from the group consisting of cytotoxic T-lymphocyte antigen 4(CTLA4), programmed cell death-1 receptor (PD-1), and programmed celldeath 1 receptor ligand (PD-L1).
 56. The method of claim 55, wherein theimmune checkpoint inhibitor is selected from the group consisting ofIpilimumab, Tremelimumab, Nivolumab, Pidilizumab, Pembrolizumab, AMP514,AUNP12, BMS-936559/MDX-1105, Atezolizumab, MPDL3280A, RG7446, R05541267,MEDI4736, Avelumab and Durvalumab.
 57. The method of claim 46, whereinthe gastrin-associated tumor and/or cancer is pancreatic cancer.
 58. Themethod of claim 57, wherein the composition induces a reduction inand/or prevents the development of fibrosis associated with thepancreatic cancer.
 59. The method of claim 46, wherein the compositionis administered in a dose selected from the group consisting of about 50μg to about 1000 μg, about 50 μg to about 500 μg, about 100 μg to about1000 μg, about 200 μg to about 1000 μg, and about 250 μg to about 500μg, and optionally wherein the dose is repeated once, twice, or threetimes, optionally wherein the second dose is administered 1 week afterthe first dose and the third dose, if administered, is administered 1 or2 weeks after the second dose.
 60. A method for inducing and/orenhancing a cellular immune response against a gastrin-associated tumoror cancer in a subject, the method comprising administering to a subjectthat has a gastrin-associated tumor or cancer an effective amount of acomposition comprising an agent that reduces or inhibits gastrinsignaling via CCK-B receptors present on a gastrin-associated tumor orcancer.
 61. The method of claim 60, wherein the agent comprises agastrin peptide, an anti-gastrin antibody, an anti-CCK-B receptorantibody, or any combination thereof.
 62. The method of claim 61,wherein the gastrin peptide comprises a gastrin-17 (G17) peptide or animmunogenic fragment thereof.
 63. The method of claim 62, wherein thegastrin peptide or the immunogenic fragment thereof comprises, consistsessentially of, or consists of an amino acid sequence selected from thegroup consisting of EGPWLEEEEE (SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2),EGPWLEEEEEAY (SEQ ID NO: 3), and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4). 64.The method of claim 61, wherein the agent comprises a gastrin peptideconjugated to an immunogenic carrier, optionally wherein the immunogeniccarrier is selected from the group consisting of diphtheria toxoid,tetanus toxoid, keyhole limpet hemocyanin, and bovine serum albumin. 65.The method of claim 64, wherein the gastrin peptide is conjugated to theimmunogenic carrier via a linker, optionally a linker comprising aε-maleimido caproic acid N-hydroxysuccinamide ester.
 66. The method ofclaim 64, wherein the linker and the gastrin peptide are separated by anamino acid spacer, optionally wherein the amino acid spacer is between 1and 10 amino acids in length, further optionally wherein the amino acidspacer is 7 amino acids in length.
 67. The method of claim 60, whereinthe composition further comprises an adjuvant, optionally an oil-basedadjuvant.
 68. A method for sensitizing a tumor and/or a cancerassociated with gastrin and/or CCK-B receptor signaling in a subject toan inducer of a cellular immune response directed against the tumorand/or cancer, the method comprising administering to the subject acomposition comprising: (a) a first agent that induces and/or providesan active and/or a passive humoral immune response against a gastrinpeptide, and a second agent that induces and/or provides a cellularimmune response against the tumor and/or the cancer, or a combinationthereof, optionally wherein the first agent and the second agent areindividually selected from the group consisting of a gastrin peptideand/or a fragment and/or a derivative thereof that induces a cellularimmune response or production of neutralizing anti-gastrin antibodies inthe subject and a neutralizing anti-gastrin antibody and/or a fragmentand/or derivative thereof and; and/or (b) a composition comprising anucleic acid that inhibits expression of a gastrin gene product; and/or(c) a composition comprising an agent that blocks the biologicalfunction of gastrin at the CCK-B receptor.
 69. The method of claim 68,wherein the anti-gastrin antibody is an antibody directed against anepitope present within gastrin-17 (G17).
 70. The method of claim 69,wherein the epitope is present within the amino acid sequence EGPWLEEEEE(SEQ ID NO: 1) or EGPWLEEEE (SEQ ID NO: 2) or EGPWLEEEEEAY (SEQ ID NO:3) OR EGPWLEEEEEAYGWMDF (SEQ ID NO: 4).
 71. The method of any of claims68-70, wherein the composition comprises the gastrin peptide conjugatedto an immunogenic carrier.
 72. The method of claim 71, wherein thegastrin peptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.73. The method of claim 71, wherein the immunogenic carrier is selectedfrom the group consisting of diphtheria toxoid, tetanus toxoid, keyholelimpet hemocyanin, and bovine serum albumin.
 74. The method of claim 71,wherein the gastrin peptide is conjugated to the immunogenic carrier viaa linker.
 75. The method of claim 74, wherein the linker comprises aε-maleimido caproic acid N-hydroxysuccinamide ester.
 76. The method ofclaim 74 or claim 75, wherein the linker and the gastrin peptide areseparated by an amino acid spacer, optionally wherein the amino acidspacer is between 1 and 10 amino acids in length, further optionallywherein the amino acid spacer is 7 amino acids in length.
 77. The methodof any of claims 68-76, wherein the composition further comprises anadjuvant, optionally an oil-based adjuvant.
 78. The method of any ofclaims 68-77, wherein the inducer of the cellular immune responseagainst the gastrin-associated tumor and/or cancer comprises an immunecheckpoint inhibitor.
 79. The method of claim 78, wherein the immunecheckpoint inhibitor inhibits a biological activity of a targetpolypeptide selected from the group consisting of cytotoxic T-lymphocyteantigen 4 (CTLA4), programmed cell death-1 receptor (PD-1), andprogrammed cell death 1 receptor ligand (PD-L1).
 80. The method of claim79, wherein the immune checkpoint inhibitor is selected from the groupconsisting of Ipilimumab, Tremelimumab, Nivolumab, Pidilizumab,Pembrolizumab, AMP514, AUNP12, BMS-936559/MDX-1105, Atezolizumab,MPDL3280A, RG7446, R05541267, MEDI4736, Avelumab and Durvalumab.
 81. Themethod of claim 68, wherein the gastrin-associated tumor and/or canceris pancreatic cancer.
 82. The method of claim 81, wherein thecomposition induces a reduction in and/or prevents the development offibrosis associated with the pancreatic cancer.
 83. The method of claim68, wherein the composition is administered in a dose selected from thegroup consisting of about 50 μg to about 1000 μg, about 50 μg to about500 μg, about 100 μg to about 1000 μg, about 200 μg to about 1000 μg,and about 250 μg to about 500 μg, and optionally wherein the dose isrepeated once, twice, or three times, optionally wherein the second doseis administered 1 week after the first dose and the third dose, ifadministered, is administered 1 or 2 weeks after the second dose.
 84. Amethod for preventing, reducing, and/or eliminating formation offibrosis associated with a tumor and/or a cancer, the method comprisingcontacting cells of the tumor and/or the cancer with an agent thatdirectly or indirectly inhibits one or more biological activities ofgastrin in the tumor and/or cancer.
 85. The method of claim 84, whereinthe agent: (i) provides and/or induces a humoral immune response againsta gastrin peptide, optionally wherein the agent is selected from thegroup consisting of an anti-gastrin antibody, and/or a fragment and/orderivative thereof, and a gastrin peptide that induces production ofneutralizing anti-gastrin antibodies in the subject; and/or (ii)comprises a nucleic acid that inhibits expression of a gastrin geneproduct; and/or (iii) comprises a small molecule compound that blocksthe function of the gastrin hormone.
 86. The method of claim 85, whereinthe anti-gastrin antibody is an antibody directed against an epitopepresent within gastrin-17 (G17).
 87. The method of claim 86, wherein theepitope is present within the amino acid sequence EGPWLEEEEE (SEQ ID NO:1), EGPWLEEEE (SEQ ID NO: 2), EGPWLEEEEEAY (SEQ ID NO: 3), orEGPWLEEEEEAYGWMDF (SEQ ID NO: 4).
 88. The method of any one of claims84-87, wherein the agent comprises the gastrin peptide that inducesproduction of neutralizing anti-gastrin antibodies conjugated to animmunogenic carrier.
 89. The method of claim 88, wherein the gastrinpeptide comprises an amino acid sequence selected from the groupconsisting of EGPWLEEEEE (SEQ ID NO: 1), EGPWLEEEE (SEQ ID NO: 2),EGPWLEEEEEAY (SEQ ID NO: 3), and EGPWLEEEEEAYGWMDF (SEQ ID NO: 4). 90.The method of claim 88, wherein the immunogenic carrier is selected fromthe group consisting of diphtheria toxoid, tetanus toxoid, keyholelimpet hemocyanin, and bovine serum albumin.
 91. The method of claim 88,wherein the gastrin peptide is conjugated to the immunogenic carrier viaa linker.
 92. The method of claim 91, wherein the linker comprises aε-maleimido caproic acid N-hydroxysuccinamide ester.
 93. The method ofclaim 91 or claim 92, wherein the linker and the gastrin peptide areseparated by an amino acid spacer, optionally wherein the amino acidspacer is between 1 and 10 amino acids in length, further optionallywherein the amino acid spacer is 7 amino acids in length.
 94. The methodof any one of claims 84-93, wherein the agent further comprises anadjuvant, optionally an oil-based adjuvant.
 95. The method of any one ofclaims 84-94, further comprising contacting the tumor and/or the cancerwith a second agent comprising a stimulator of a cellular immuneresponse against the tumor and/or the cancer.
 96. The method of claim95, wherein the second agent is an immune checkpoint inhibitor.
 97. Themethod of claim 96, wherein the immune checkpoint inhibitor inhibits abiological activity of a target polypeptide selected from the groupconsisting of cytotoxic T-lymphocyte antigen 4 (CTLA4), programmed celldeath-1 receptor (PD-1), and programmed cell death 1 receptor ligand(PD-L1).
 98. The method of claim 97, wherein the immune checkpointinhibitor is selected from the group consisting of Ipilimumab,Tremelimumab, Nivolumab, Pidilizumab, Pembrolizumab, AMP514, AUNP12,BMS-936559/MDX-1105, Atezolizumab, MPDL3280A, RG7446, R05541267,MEDI4736, and Avelumab.
 99. The method of any one of claims 84-98,wherein the tumor and/or cancer is pancreatic cancer.
 100. Use of thepharmaceutical composition of any one of claims 1-13 for preventing,reducing, and/or eliminating metastasis of a gastrin-associated tumor orcancer by administering to a subject having a gastrin-associated tumoror cancer an amount of the pharmaceutical composition of any one ofclaims 1-13 sufficient to enhance the number of CD4⁻/CD8⁻ T_(EMRA) cellsin the subject that respond to the gastrin-associated tumor or cancer.101. Use of the pharmaceutical composition of any one of claims 1-13 forincreasing the number of T_(EMRA) cells in a subject that respond to agastrin-associated tumor or cancer.
 102. Use of a composition comprisingan immune checkpoint inhibitor and a gastrin immunogen to treat agastrin-associated tumor or cancer.
 103. Use of a composition comprisingan immune checkpoint inhibitor and a gastrin immunogen for thepreparation of a medicament to treat a gastrin-associated tumor orcancer.
 104. Use of the pharmaceutical composition of any one of claims1-13 for preventing, reducing, and/or eliminating metastasis of agastrin-associated tumor or cancer by administering to a subject havinga gastrin-associated tumor or cancer an amount of the pharmaceuticalcomposition of any one of claims 1-13 sufficient to enhance the numberof CD8⁺ tumor infiltrating lymphocytes.
 105. The use of claim 104,wherein the administering results in improves survival of the subject,reduced tumor growth, and/or enhanced efficacy of a chemotherapeuticagent and/or an immune checkpoint therapy in the subject as compared tothat which would have occurred had the pharmaceutical composition notbeen administered.
 106. Use of the pharmaceutical composition of any oneof claims 1-13 for preventing, reducing, and/or eliminating metastasisof a gastrin-associated tumor or cancer by administering to a subjecthaving a gastrin-associated tumor or cancer an amount of thepharmaceutical composition of any one of claims 1-13 sufficient toreduce the number of FoxP3⁺ inhibitory T-regulatory cells.